Maize hybrid X00N540

ABSTRACT

A novel maize variety designated X00N540 and seed, plants and plant parts thereof are produced by crossing inbred maize varieties. Methods for producing a maize plant by crossing hybrid maize variety X00N540 with another maize plant are disclosed. Methods for producing a maize plant containing in its genetic material one or more traits introgressed into X00N540 through backcrossing or genetic transformation, and to the maize seed, plant and plant part produced thereby are described. Maize variety X00N540, the seed, the plant produced from the seed, and variants, mutants, and minor modifications of maize variety X00N540 are provided. Methods for producing maize varieties derived from maize variety X00N540 and methods of using maize variety X00N540 are disclosed.

BACKGROUND

The goal of hybrid development is to combine, in a single hybrid,various desirable traits. For field crops, these traits may includeresistance to diseases and insects, resistance to heat and drought,reducing the time to crop maturity, greater yield, and better agronomicquality. With mechanical harvesting of many crops, uniformity of plantcharacteristics such as germination, stand establishment, growth rate,maturity, and plant and ear height is important. Traditional plantbreeding is an important tool in developing new and improved commercialcrops.

SUMMARY

Provided is a novel maize, Zea mays L., variety, seed, plant, cells andits parts designated as X00N540, produced by crossing two maize inbredvarieties. The hybrid maize variety X00N540, the seed, the plant and itsparts produced from the seed, and variants, mutants and minormodifications of maize X00N540 are provided. Processes are provided formaking a maize plant containing in its genetic material one or moretraits introgressed into X00N540 through locus conversion, backcrossingand/or transformation, and to the maize seed, plant and plant partsproduced thereby. Methods for producing maize varieties derived fromhybrid maize variety X00N540 are also provided. Also provided are maizeplants having all the physiological and morphological characteristics ofthe hybrid maize variety X00N540.

The hybrid maize plant may further comprise a cytoplasmic or nuclearfactor capable of conferring male sterility or otherwise preventingself-pollination, such as by self-incompatibility. Parts of the maizeplants disclosed herein are also provided, for example, pollen obtainedfrom a hybrid plant and an ovule of the hybrid plant.

Seed of the hybrid maize variety X00N540 is provided and may be providedas a population of maize seed of the variety designated X00N540.

Compositions are provided comprising a seed of maize variety X00N540comprised in plant seed growth media. In certain embodiments, the plantseed growth media is a soil or synthetic cultivation medium. In specificembodiments, the growth medium may be comprised in a container or may,for example, be soil in a field. Hybrid maize variety X00N540 isprovided comprising an added heritable trait.

The heritable trait may be a genetic locus that is a dominant orrecessive allele. In certain embodiments, the genetic locus conferstraits such as, for example, male sterility, waxy starch, herbicidetolerance or resistance, insect resistance, resistance to bacterial,fungal, nematode or viral disease, and altered or modified fatty acid,phytate, protein or carbohydrate metabolism. The genetic locus may be anaturally occurring maize gene introduced into the genome of a parent ofthe variety by backcrossing, a natural or induced mutation, or atransgene introduced through genetic transformation techniques. Whenintroduced through transformation, a genetic locus may comprise one ormore transgenes integrated at a single chromosomal location.

A hybrid maize plant of the variety designated X00N540 is provided,wherein a cytoplasmically-inherited trait has been introduced into thehybrid plant. Such cytoplasmically-inherited traits are passed toprogeny through the female parent in a particular cross. An exemplarycytoplasmically-inherited trait is the male sterility trait.Cytoplasmic-male sterility (CMS) is a pollen abortion phenomenondetermined by the interaction between the genes in the cytoplasm and thenucleus. Alteration in the mitochondrial genome and the lack of restorergenes in the nucleus will lead to pollen abortion. With either a normalcytoplasm or the presence of restorer gene(s) in the nucleus, the plantwill produce pollen normally. A CMS plant can be pollinated by amaintainer version of the same variety, which has a normal cytoplasm butlacks the restorer gene(s) in the nucleus, and continues to be malesterile in the next generation. The male fertility of a CMS plant can berestored by a restorer version of the same variety, which must have therestorer gene(s) in the nucleus. With the restorer gene(s) in thenucleus, the offspring of the male-sterile plant can produce normalpollen grains and propagate. A cytoplasmically inherited trait may be anaturally occurring maize trait or a trait introduced through genetictransformation techniques.

A tissue culture of regenerable cells of a plant of variety X00N540 isprovided. The tissue culture can be capable of regenerating plantscapable of expressing all of the physiological and morphological orphenotypic characteristics of the variety and of regenerating plantshaving substantially the same genotype as other plants of the variety.Examples of some of the physiological and morphological characteristicsof the variety X00N540 that may be assessed include characteristicsrelated to yield, maturity, and kernel quality. The regenerable cells insuch tissue cultures can be derived, for example, from embryos,meristematic cells, immature tassels, microspores, pollen, leaves,anthers, roots, root tips, silk, flowers, kernels, ears, cobs, husks, orstalks, or from callus or protoplasts derived from those tissues. Maizeplants regenerated from the tissue cultures and plants having all oressentially all of the physiological and morphological characteristicsof variety X00N540 are also provided.

A method of producing hybrid maize seed comprising crossing a plant ofvariety PH48RS with a plant of variety PH42V3. In a cross, either parentmay serve as the male or female. Processes are also provided forproducing maize seeds or plants, which processes generally comprisecrossing a first parent maize plant as a male or female parent with asecond parent maize plant, wherein at least one of the first or secondparent maize plants is a plant of the variety designated X00N540. Insuch crossing, either parent may serve as the male or female parent.These processes may be further exemplified as processes for preparinghybrid maize seed or plants, wherein a first hybrid maize plant iscrossed with a second maize plant of a different, distinct variety toprovide a hybrid that has, as one of its parents, the hybrid maize plantvariety X00N540. In these processes, crossing will result in theproduction of seed. The seed production occurs regardless of whether theseed is collected or not.

In some embodiments, the first step in “crossing” comprises planting,often in pollinating proximity, seeds of a first and second parent maizeplant, and in many cases, seeds of a first maize plant and a second,distinct maize plant. Where the plants are not in pollinating proximity,pollination can nevertheless be accomplished by other means, such as bytransferring a pollen or tassel bag from one plant to the other.

A second step comprises cultivating or growing the seeds of said firstand second parent maize plants into plants that bear flowers (maizebears both male flowers (tassels) and female flowers (silks) in separateanatomical structures on the same plant).

A third step comprises preventing self-pollination of the plants, i.e.,preventing the silks of a plant from being fertilized by any plant ofthe same variety, including the same plant. This can be done, forexample, by emasculating the male flowers of the first or second parentmaize plant, (i.e., treating or manipulating the flowers so as toprevent pollen production, in order to produce an emasculated parentmaize plant). Self-incompatibility systems may also be used in somehybrid crops for the same purpose. Self-incompatible plants still shedviable pollen and can pollinate plants of other varieties but areincapable of pollinating themselves or other plants of the same variety.

A fourth step may comprise allowing cross-pollination to occur betweenthe first and second parent maize plants. When the plants are not inpollinating proximity, this can be done by placing a bag, usually paperor glassine, over the tassels of the first plant and another bag overthe silks of the incipient ear on the second plant. The bags are left inplace for at least 24 hours. Since pollen is viable for less than 24hours, this assures that the silks are not pollinated from other pollensources, that any stray pollen on the tassels of the first plant isdead, and that the only pollen transferred comes from the first plant.The pollen bag over the tassel of the first plant is then shakenvigorously to enhance release of pollen from the tassels, and the shootbag is removed from the silks of the incipient ear on the second plant.Finally, the pollen bag is removed from the tassel of the first plantand is placed over the silks of the incipient ear of the second plant,shaken again and left in place. Yet another step comprises harvestingthe seeds from at least one of the parent maize plants. The harvestedseed can be grown to produce a maize plant or hybrid maize plant.

Maize seed and plants are provided that are produced by a process thatcomprises crossing a first parent maize plant with a second parent maizeplant, wherein at least one of the first or second parent maize plantsis a plant of the variety designated X00N540. Maize seed and plantsproduced by the process are first generation hybrid maize seed andplants produced by crossing an inbred with another, distinct inbred.Seed of an F1 hybrid maize plant, an F1 hybrid maize plant and seedthereof, specifically the hybrid variety designated X00N540 is provided.

Plants described herein can be analyzed by their “genetic complement.”This term is used to refer to the aggregate of nucleotide sequences, theexpression of which defines the phenotype of, for example, a maizeplant, or a cell or tissue of that plant. A genetic complement thusrepresents the genetic makeup of a cell, tissue or plant. Provided aremaize plant cells that have a genetic complement in accordance with themaize plant cells disclosed herein, and plants, seeds and diploid plantscontaining such cells.

Plant genetic complements may be assessed by genetic marker profiles,and by the expression of phenotypic traits that are characteristic ofthe expression of the genetic complement, e.g., isozyme typing profiles.It is understood that variety X00N540 could be identified by any of themany well-known techniques used for genetic profiling disclosed herein.

DETAILED DESCRIPTION

A new and distinctive maize hybrid variety designated X00N540, which hasbeen the result of years of careful breeding and selection in acomprehensive maize breeding program is provided.

Definitions

Maize, Zea mays L., can be referred to as maize or corn. Certaindefinitions used in the specification are provided below. Also in theexamples that follow, a number of terms are used herein. In order toprovide a clear and consistent understanding of the specification andclaims, including the scope to be given such terms, the followingdefinitions are provided. NOTE: ABS is in absolute terms and % MN ispercent of the mean for the experiments in which the inbred or hybridwas grown. PCT designates that the trait is calculated as a percentage.% NOT designates the percentage of plants that did not exhibit a trait.For example, STKLDG % NOT is the percentage of plants in a plot thatwere not stalk lodged. These designators will follow the descriptors todenote how the values are to be interpreted. Below are the descriptorsused in the data tables included herein.

BRITTLE STALK: A count of the number of “snapped” plants per plotfollowing machine snapping or artificial selection pressure. A snappedplant has its stalk completely snapped at a node between the base of theplant and the node above the ear. Can be expressed as percent of plantsthat did not snap.

ALLELE: Any of one or more alternative forms of a genetic sequence. In adiploid cell or organism, the two alleles of a given sequence typicallyoccupy corresponding loci on a pair of homologous chromosomes.

ALTER: With respect to genetic manipulation, the utilization ofup-regulation, down-regulation, or gene silencing.

ANTHESIS: The time of a flower's opening.

ANTHRACNOSE STALK ROT (Colletotrichum graminicola): A 1 to 9 visualrating indicating the resistance to Anthracnose Stalk Rot. A higherscore indicates a higher resistance. Data are collected only whensufficient selection pressure exists in the experiment measured.

BLUP=BEST LINEAR UNBIASED PREDICTION. The BLUP values are determinedfrom a mixed model analysis of hybrid performance observations atvarious locations and replications. BLUP values for inbred maize plants,breeding values, are estimated from the same analysis using pedigreeinformation.

BREEDING CROSS: A cross to introduce new genetic material into a plantfor the development of a new variety. For example, one could cross plantA with plant B, wherein plant B would be genetically different fromplant A. After the breeding cross, the resulting F1 plants could then beselfed or sibbed for one, two, three or more times (F1, F2, F3, etc.)until a new inbred variety is developed.

CELL: Cell as used herein includes a plant cell, whether isolated, intissue culture or incorporated in a plant or plant part.

CORN LETHAL NECROSIS: Synergistic interaction of maize chlorotic mottlevirus (MCMV) in combination with either maize dwarf mosaic virus (MDMV-Aor MDMV-B) or wheat streak mosaic virus (WSMV). A 1 to 9 visual ratingindicating the resistance to Corn Lethal Necrosis. A higher scoreindicates a higher resistance. Data are collected only when sufficientselection pressure exists in the experiment measured.

COMMON SMUT: This is the percentage of plants not infected with CommonSmut. Data are collected only when sufficient selection pressure existsin the experiment measured.

COMMON RUST (Puccinia sorghi): A 1 to 9 visual rating indicating theresistance to Common Rust. A higher score indicates a higher resistance.Data are collected only when sufficient selection pressure exists in theexperiment measured.

CROSS POLLINATION: Fertilization by the union of two gametes fromdifferent plants.

CROSSING: The combination of genetic material by traditional methodssuch as a breeding cross or backcross, but also including protoplastfusion and other molecular biology methods of combining genetic materialfrom two sources.

D and D1-Dn: represents the generation of doubled haploid.

DRYDOWN: This represents the relative rate at which a hybrid will reachacceptable harvest moisture compared to other hybrids on a 1 to 9 ratingscale. A high score indicates a hybrid that dries relatively fast whilea low score indicates a hybrid that dries slowly.

DIGESTIBLE ENERGY: Near-infrared transmission spectroscopy, NIT,prediction of digestible energy.

DIPLODIA EAR MOLD SCORES (Diplodia maydis and Diplodia macrospora): A 1to 9 visual rating indicating the resistance to Diplodia Ear Mold. Ahigher score indicates a higher resistance. Data are collected only whensufficient selection pressure exists in the experiment measured.

DIPLODIA STALK ROT: Stalk rot severity due to Diplodia (Diplodiamaydis). Expressed as a 1 to 9 score with 9 being highly resistant. Dataare collected only when sufficient selection pressure exists in theexperiment measured.

DROPPED EARS: A measure of the number of dropped ears per plot andrepresents the percentage of plants that did not drop ears prior toharvest. Data are collected only when sufficient selection pressureexists in the experiment measured.

DROUGHT TOLERANCE: This represents a 1 to 9 rating for droughttolerance, and is based on data obtained under stress conditions. A highscore indicates good drought tolerance and a low score indicates poordrought tolerance. Data are collected only when sufficient selectionpressure exists in the experiment measured.

EYE SPOT (Kabatiella zeae or Aureobasidium zeae): A 1 to 9 visual ratingindicating the resistance to Eye Spot. A higher score indicates a higherresistance. Data are collected only when sufficient selection pressureexists in the experiment measured.

F1 PROGENY: A progeny plant produced by crossing a plant of one maizeline with a plant of another maize line.

FUSARIUM EAR ROT (Fusarium moniliforme or Fusarium subglutinans): A 1 to9 visual rating indicating the resistance to Fusarium Ear Rot. A higherscore indicates a higher resistance. Data are collected only whensufficient selection pressure exists in the experiment measured.

GDU=GROWING DEGREE UNITS: Using the Barger Heat Unit Theory, whichassumes that maize growth occurs in the temperature range 50° F.-86° F.and that temperatures outside this range slow down growth; the maximumdaily heat unit accumulation is 36 and the minimum daily heat unitaccumulation is 0. The seasonal accumulation of GDU is a major factor indetermining maturity zones.

GDUSHD=GDU TO SHED: The number of growing degree units (GDUs) or heatunits required for an inbred variety or hybrid to have approximately 50percent of the plants shedding pollen and is measured from the time ofplanting. Growing degree units are calculated by the Barger Method,where the heat units for a 24-hour period are:

${G\; D\; U} = {\frac{( {{Max}.\mspace{14mu}{temp}.{+ {{Min}.\mspace{14mu}{temp}.}}} )}{2} - 50}$

The units determined by the Barger Method are then divided by 10. Thehighest maximum temperature used is 86 degrees F. and the lowest minimumtemperature used is 50 degrees F. For each inbred or hybrid it takes acertain number of GDUs to reach various stages of plant development.

GDUSLK=GDU TO SILK: The number of growing degree units required for aninbred variety or hybrid to have approximately 50 percent of the plantswith silk emergence from time of planting. Growing degree units arecalculated by the Barger Method as given in GDUSHD definition and thendivided by 10.

GENE SILENCING: The interruption or suppression of the expression of agene at the level of transcription or translation.

GENOTYPE: Refers to the genetic mark-up or profile of a cell ororganism.

GIBERS=GIBBERELLA EAR ROT (PINK MOLD) (Gibberella zeae): A 1 to 9 visualrating indicating the resistance to Gibberella Ear Rot. A higher scoreindicates a higher resistance. Data are collected only when sufficientselection pressure exists in the experiment measured.

GIBROT=GIBBERELLA STALK ROT SCORE: Score of stalk rot severity due toGibberella (Gibberella zeae). Expressed as a 1 to 9 score with 9 beinghighly resistant. Data are collected only when sufficient selectionpressure exists in the experiment measured.

GLFSPT=GRAY LEAF SPOT (Cercospora zeae-maydis): A 1 to 9 visual ratingindicating the resistance to Gray Leaf Spot. A higher score indicates ahigher resistance. Data are collected only when sufficient selectionpressure exists in the experiment measured.

GOSWLT=GOSS' WILT (Corynebacterium nebraskense): A 1 to 9 visual ratingindicating the resistance to Goss' Wilt. A higher score indicates ahigher resistance. Data are collected only when sufficient selectionpressure exists in the experiment measured.

GRAIN TEXTURE: A visual rating used to indicate the appearance of maturegrain observed in the middle third of the uppermost ear when welldeveloped. Grain or seed with a hard grain texture is indicated asflint; grain or seed with a soft grain texture is indicted as dent.Medium grain or seed texture may be indicated as flint-dent orintermediate. Other grain textures include flint-like, dent-like, sweet,pop, waxy and flour.

GRNAPP=GRAIN APPEARANCE: This is a 1 to 9 rating for the generalappearance of the shelled grain as it is harvested based on such factorsas the color of harvested grain, any mold on the grain, and any crackedgrain. Higher scores indicate better grain visual quality.

H and H1: Refers to the haploid generation.

HAPLOID PLANT PART: Refers to a plant part or cell that has a haploidgenotype.

HCBLT=HELMINTHOSPORIUM CARBONUM LEAF BLIGHT (Helminthosporium carbonum):A 1 to 9 visual rating indicating the resistance to Helminthosporiuminfection. A higher score indicates a higher resistance. Data arecollected only when sufficient selection pressure exists in theexperiment measured.

HD SMT=HEAD SMUT (Sphacelotheca reiliana): This indicates the percentageof plants not infected. Data are collected only when sufficientselection pressure exists in the experiment measured.

HSKCVR=HUSK COVER: A 1 to 9 score based on performance relative to keychecks, with a score of 1 indicating very short husks, tip of ear andkernels showing; 5 is intermediate coverage of the ear under mostconditions, sometimes with thin husk; and a 9 has husks extending andclosed beyond the tip of the ear. Scoring can best be done nearphysiological maturity stage or any time during dry down untilharvested.

HTFRM=Near-infrared transmission spectroscopy, NIT, prediction offermentables.

HYBRID VARIETY: A substantially heterozygous hybrid line and minorgenetic modifications thereof that retain the overall genetics of thehybrid line.

INBRED: A variety developed through inbreeding or doubled haploidy thatpreferably comprises homozygous alleles at about 95% or more of itsloci. An inbred can be reproduced by selfing or growing in isolation sothat the plants can only pollinate with the same inbred variety.

INTROGRESSION: The process of transferring genetic material from onegenotype to another.

KERNEL PERICARP COLOR is scored when kernels have dried down and istaken at or about 65 days after 50% silk. Score codes are: Colorless=1;Red with white crown=2; Tan=3; Bronze=4; Brown=5; Light red=6; Cherryred=7.

KER_WT=KERNEL NUMBER PER UNIT WEIGHT (Pounds or Grams): The number ofkernels in a specific measured weight; determined after removal ofextremely small and large kernels.

LINKAGE: Refers to a phenomenon wherein alleles on the same chromosometend to segregate together more often than expected by chance if theirtransmission was independent.

LINKAGE DISEQUILIBRIUM: Refers to a phenomenon wherein alleles tend toremain together in linkage groups when segregating from parents tooffspring, with a greater frequency than expected from their individualfrequencies.

LOCUS: A specific location on a chromosome.

LOCUS CONVERSION: (Also called TRAIT CONVERSION) A locus conversionrefers to plants within a variety that have been modified in a mannerthat retains the overall genetics of the variety and further comprisesone or more loci with a specific desired trait, such as male sterility,insect resistance, disease resistance or herbicide tolerance orresistance. Examples of single locus conversions include mutant genes,transgenes and native traits finely mapped to a single locus. One ormore locus conversion traits may be introduced into a single cornvariety.

LRTLPN=LATE ROOT LODGING: An estimate of the percentage of plants thatdo not root lodge after anthesis through harvest; plants that lean fromthe vertical axis at an approximately 30-degree angle or greater wouldbe considered as root lodged. Data are collected only when sufficientselection pressure exists in the experiment measured.

LRTLSC=LATE ROOT LODGING SCORE: Score for severity of plants that leanfrom a vertical axis at an approximate 30-degree angle or greater whichtypically results from strong winds after flowering. Recorded prior toharvest when a root-lodging event has occurred. This lodging results inplants that are leaned or “lodged” over at the base of the plant and donot straighten or “goose-neck” back to a vertical position. Expressed asa 1 to 9 score with 9 being no lodging. Data are collected only whensufficient selection pressure exists in the experiment measured.

MALE STERILITY: A male sterile plant is one which produces no viablepollen no (pollen that is able to fertilize the egg to produce a viableseed). Male sterility prevents self pollination. These male sterileplants are therefore useful in hybrid plant production.

MDMCPX=MAIZE DWARF MOSAIC COMPLEX (MDMV=Maize Dwarf Mosaic Virus andMCDV=Maize Chlorotic Dwarf Virus). A 1 to 9 visual rating indicating theresistance to Maize Dwarf Mosaic Complex. A higher score indicates ahigher resistance. Data are collected only when sufficient selectionpressure exists in the experiment measured.

MILKLN=percent milk in mature grain.

MST=HARVEST MOISTURE: The moisture is the actual percentage moisture ofthe grain at harvest.

NEI DISTANCE: A quantitative measure of percent similarity between twovarieties. Nei's distance between varieties A and B can be defined as1−(2*number alleles in common/(number alleles in A+number alleles in B).For example, if varieties A and B are the same for 95 out of 100alleles, the Nei distance would be 0.05. If varieties A and B are thesame for 98 out of 100 alleles, the Nei distance would be 0.02. Freesoftware for calculating Nei distance is available on the internet atmultiple locations. See Nei, Proc Natl Acad Sci, 76:5269-5273 (1979).

NLFBLT=NORTHERN LEAF BLIGHT (Helminthosporium turcicum or Exserohilumturcicum): A 1 to 9 visual rating indicating the resistance to NorthernLeaf Blight. A higher score indicates a higher resistance. Data arecollected only when sufficient selection pressure exists in theexperiment measured.

NUCLEIC ACID: An acidic, chainlike biological macromolecule consistingof multiple repeat units of phosphoric acid, sugar, and purine andpyrimidine bases.

OILT=GRAIN OIL: Absolute value of oil content of the kernel as predictedby Near-Infrared Transmittance and expressed as a percent of dry matter.

PERCENT IDENTITY: Percent identity as used herein refers to thecomparison of the alleles present in two varieties. For example, whencomparing two inbred plants to each other, each inbred plant will havethe same allele (and therefore be homozygous) at almost all of theirloci. Percent identity is determined by comparing a statisticallysignificant number of the homozygous alleles of two varieties. Forexample, a percent identity of 90% between X00N540 and other varietymeans that the two varieties have the same homozygous alleles at 90% oftheir loci.

PLANT: As used herein, the term “plant” includes reference to animmature or mature whole plant, including a plant that has beendetasseled or from which seed or grain has been removed. Seed or embryothat will produce the plant is also considered to be the plant.

PLANT PART: As used herein, the term “plant part” includes leaves,stems, roots, seed, grain, embryo, pollen, ovules, flowers, ears, cobs,husks, stalks, root tips, anthers, pericarp, silk, tissue, cells and thelike. In some embodiments, the plant part contains at least one cell ofhybrid maize variety X00N540 or a locus conversion thereof.

PLATFORM indicates the variety with the base genetics and the varietywith the base genetics comprising locus conversion(s). There can be aplatform for the inbred maize variety and the hybrid maize variety.PLTHT=PLANT HEIGHT: This is a measure of the height of the plant fromthe ground to the tip of the tassel in inches.

POLSC=POLLEN SCORE: A 0 to 9 visual rating indicating the amount ofpollen shed. The higher the score the more pollen shed.

POLWT=POLLEN WEIGHT: This is calculated by dry weight of tasselscollected as shedding commences minus dry weight from similar tasselsharvested after shedding is complete.

RM=RELATIVE MATURITY: This is a predicted relative maturity based on theharvest moisture of the grain. The relative maturity rating is based ona known set of checks and utilizes standard linear regression analysesand is also referred to as the Comparative Relative Maturity RatingSystem that is similar to the Minnesota Relative Maturity Rating System.

PROT=GRAIN PROTEIN: Absolute value of protein content of the kernel aspredicted by Near-Infrared Transmittance and expressed as a percent ofdry matter.

RESISTANCE: Synonymous with tolerance. The ability of a plant towithstand exposure to an insect, disease, herbicide or other condition.A resistant plant variety will have a level of resistance higher than acomparable wild-type variety.

ROOT LODGING: Root lodging is the percentage of plants that do not rootlodge; plants that lean from the vertical axis at an approximately30-degree angle or greater would be counted as root lodged. Data arecollected only when sufficient selection pressure exists in theexperiment measured.

SEED: Fertilized and ripened ovule, consisting of the plant embryo,varying amounts of stored food material, and a protective outer seedcoat. Synonymous with grain.

SEL IND=SELECTION INDEX: The selection index gives a single measure ofthe hybrid's worth based on information for multiple traits. A maizebreeder may utilize his or her own set of traits for the selectionindex. One of the traits that is almost always included is yield. Theselection index data presented in the tables represent the mean valueaveraged across testing stations.

SELF POLLINATION: A plant is self-pollinated if pollen from one floweris transferred to the same or another flower of the same plant.

SIB POLLINATION: A plant is sib-pollinated when individuals within thesame family or variety are used for pollination.

SITE SPECIFIC INTEGRATION: Genes that create a site for site specificDNA integration. This includes the introduction of FRT sites that may beused in the FLP/FRT system and/or Lox sites that may be used in theCre/Loxp system. For example, see Lyznik, et al., Site-SpecificRecombination for Genetic Engineering in Plants, Plant Cell Rep (2003)21:925-932 and WO 99/25821.

SLFBLT=SOUTHERN LEAF BLIGHT (Helminthosporium maydis or Bipolarismaydis): A 1 to 9 visual rating indicating the resistance to SouthernLeaf Blight. A higher score indicates a higher resistance. Data arecollected only when sufficient selection pressure exists in theexperiment measured.

SNP=SINGLE-NUCLEOTIDE POLYMORPHISM: is a DNA sequence variationoccurring when a single nucleotide in the genome differs betweenindividual plant or plant varieties. The differences can be equated withdifferent alleles, and indicate polymorphisms. A number of SNP markerscan be used to determine a molecular profile of an individual plant orplant variety and can be used to compare similarities and differencesamong plants and plant varieties.

SOURST=SOUTHERN RUST (Puccinia polysora): A 1 to 9 visual ratingindicating the resistance to Southern Rust. A higher score indicates ahigher resistance. Data are collected only when sufficient selectionpressure exists in the experiment measured.

SPKDSC=SPIKELET DENSITY SCORE: The visual 1-9 rating of how densespikelets are on the middle tassel branches. A higher score indicateshigher spikelet density.

STAGRN=STAY GREEN: Stay green is the measure of plant health near thetime of black layer formation (physiological maturity). A high scoreindicates better late-season plant health.

STKLDS=STALK LODGING SCORE: A plant is considered as stalk lodged if thestalk is broken or crimped between the ear and the ground. This can becaused by any or a combination of the following: strong winds late inthe season, disease pressure within the stalks, ECB damage orgenetically weak stalks. This trait should be taken just prior to or atharvest. Expressed on a 1 to 9 scale with 9 being no lodging. Data arecollected only when sufficient selection pressure exists in theexperiment measured.

STLLPN=LATE STALK LODGING: This is the percent of plants that did notstalk lodge (stalk breakage or crimping) at or around late seasonharvest (when grain moisture is below 20%) as measured by either naturallodging or pushing the stalks and determining the percentage of plantsthat break or crimp below the ear. Data are collected only whensufficient selection pressure exists in the experiment measured.

STLPCN=STALK LODGING REGULAR: This is an estimate of the percentage ofplants that did not stalk lodge (stalk breakage) at regular harvest(when grain moisture is between about 20% and 30%) as measured by eithernatural lodging or pushing the stalks and determining the percentage ofplants that break below the ear. Data are collected only when sufficientselection pressure exists in the experiment measured.

STWWLT=Stewart's Wilt (Erwinia stewartii): A 1 to 9 visual ratingindicating the resistance to Stewart's Wilt. A higher score indicates ahigher resistance. Data are collected only when sufficient selectionpressure exists in the experiment measured.

SSRs: Genetic markers based on polymorphisms in repeated nucleotidesequences, such as microsatellites. A marker system based on SSRs can behighly informative in linkage analysis relative to other marker systemsin that multiple alleles may be present.

TASBRN=TASSEL BRANCH NUMBER: The number of tassel branches, with anthersoriginating from the central spike.

TASSZ=TASSEL SIZE: A 1 to 9 visual rating was used to indicate therelative size of the tassel. A higher rating means a larger tassel.

TAS WT=TASSEL WEIGHT: This is the average weight of a tassel (grams)just prior to pollen shed.

TILLER=TILLERS: A count of the number of tillers per plot that couldpossibly shed pollen was taken. Data are given as a percentage oftillers: number of tillers per plot divided by number of plants perplot. A tiller is defined as a secondary shoot that has developed as atassel capable of shedding pollen.

TSTWT=TEST WEIGHT (ADJUSTED): The measure of the weight of the grain inpounds for a given volume (bushel), adjusted for MST less than or equalto 22%.

TSTWTN=TEST WEIGHT (UNADJUSTED): The measure of the weight of the grainin pounds for a given volume (bushel).

VARIETY: A maize line and minor genetic modifications thereof thatretain the overall genetics of the line including but not limited to alocus conversion, a mutation, or a somoclonal variant.

YIELD BU/A=YIELD (BUSHELS/ACRE): Yield of the grain at harvest by weightor volume (bushels) per unit area (acre) adjusted to 15% moisture. Theyield platform BLUP is a value derived by averaging for all members ofthe platform weighted by the inverse of the Standard Errors.

YLDSC=YIELD SCORE: A 1 to 9 visual rating was used to give a relativerating for yield based on plot ear piles. The higher the rating thegreater visual yield appearance.

YIELDS=Silage Dry Matter Yield (tons/acre @ 100% DM)

MLKYLD=Estimated pounds of milk produced per ton of dry matter fed andis based on utilizing nutrient content and fiber digestibilityADJMLK=Estimated pounds of milk produced per acre of silage dry matterbased on an equation and is MLKYLD divided by YIELDS.

SLGPRM=Silage Predicted Relative Maturity Silage Yields (Tonnage)Adjusted to 30% Dry Matter PCTMST=Silage Harvest Moisture %

NDFDR=Silage Fiber Digestibility Based on rumen fluid NIRS calibration

NDFDC=Silage Fiber Digestibility Based on rumen fluid NIRS calibration

All tables discussed in the Detailed Description section can be found atthe end of the section.

Phenotypic Characteristics of X00N540

Pioneer Brand Hybrid Maize Variety X00N540 is a single cross maizevariety and can be made by crossing inbreds PH48RS and PH42V3. Locusconversions of Hybrid Maize Variety X00N540 can be made by crossinginbreds PH48RS and PH42V3 wherein PH48RS and/or PH42V3 comprise a locusconversion(s).

The maize variety has shown uniformity and stability within the limitsof environmental influence for all the traits as described in theVariety Description Information (see Table 1, found at the end of thesection). The inbred parents of this maize variety have beenself-pollinated and ear-rowed a sufficient number of generations withcareful attention paid to uniformity of plant type to ensure thehomozygosity and phenotypic stability necessary for use in commercialhybrid seed production. The variety has been increased both by hand andin isolated fields with continued observation for uniformity. No varianttraits have been observed or are expected in X00N540.

Hybrid Maize Variety X00N540 can be reproduced by planting seeds of theinbred parent varieties, growing the resulting maize plants under crosspollinating conditions, and harvesting the resulting seed usingtechniques familiar to the agricultural arts.

Genotypic Characteristics of X00N540

In addition to phenotypic observations, a plant can also be described oridentified by its genotype. The genotype of a plant can be characterizedthrough a genetic marker profile. Genetic marker profiles can beobtained by techniques such as Restriction Fragment Length Polymorphisms(RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily PrimedPolymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting(DAF), Sequence Characterized Amplified Regions (SCARs), AmplifiedFragment Length Polymorphisms (AFLPs), Simple Sequence Repeats (SSRs)which are also referred to as Microsatellites, and Single NucleotidePolymorphisms (SNPs). For example, see Berry et al. (2002), “AssessingProbability of Ancestry Using Simple Sequence Repeat Profiles:Applications to Maize Hybrids and Inbreds”, Genetics 161: 813-824, andBerry et al. (2003), “Assessing Probability of Ancestry Using SimpleSequence Repeat Profiles: Applications to Maize Inbred Lines and SoybeanVarieties”, Genetics 165: 331-342.

Particular markers used for these purposes may include any type ofmarker and marker profile which provides a means of distinguishingvarieties. A genetic marker profile can be used, for example, toidentify plants of the same variety or related varieties or to determineor validate a pedigree. In addition to being used for identification ofmaize variety X00N540 and its plant parts, the genetic marker profile isalso useful in developing a locus conversion of X00N540.

Methods of isolating nucleic acids from maize plants and methods forperforming genetic marker profiles using SNP and SSR polymorphisms arewell known in the art. SNPs are genetic markers based on a polymorphismin a single nucleotide. A marker system based on SNPs can be highlyinformative in linkage analysis relative to other marker systems in thatmultiple alleles may be present.

A method comprising isolating nucleic acids, such as DNA, from a plant,a plant part, plant cell or a seed of the maize plants disclosed hereinis provided. The method can include mechanical, electrical and/orchemical disruption of the plant, plant part, plant cell or seed,contacting the disrupted plant, plant part, plant cell or seed with abuffer or solvent, to produce a solution or suspension comprisingnucleic acids, optionally contacting the nucleic acids with aprecipitating agent to precipitate the nucleic acids, optionallyextracting the nucleic acids, and optionally separating the nucleicacids such as by centrifugation or by binding to beads or a column, withsubsequent elution, or a combination thereof. If DNA is being isolated,an RNase can be included in one or more of the method steps. The nucleicacids isolated can comprise all or substantially all of the genomic DNAsequence, all or substantially all of the chromosomal DNA sequence orall or substantially all of the coding sequences (cDNA) of the plant,plant part, or plant cell from which they were isolated. The amount andtype of nucleic acids isolated may be sufficient to permit whole genomesequencing of the plant from which they were isolated or chromosomalmarker analysis of the plant from which they were isolated.

The methods can be used to produce nucleic acids from the plant, plantpart, seed or cell, which nucleic acids can be, for example, analyzed toproduce data. The data can be recorded. The nucleic acids from thedisrupted cell, the disrupted plant, plant part, plant cell or seed orthe nucleic acids following isolation or separation can be contactedwith primers and nucleotide bases, and/or a polymerase to facilitate PCRsequencing or marker analysis of the nucleic acids. In some examples,the nucleic acids produced can be sequenced or contacted with markers toproduce a genetic profile, a molecular profile, a marker profile, ahaplotype, or any combination thereof. In some examples, the geneticprofile or nucleotide sequence is recorded on a computer readablemedium. In other examples, the methods may further comprise using thenucleic acids produced from plants, plant parts, plant cells or seeds ina plant breeding program, for example in making crosses, selectionand/or advancement decisions in a breeding program. Crossing includesany type of plant breeding crossing method, including but not limited tocrosses to produce hybrids, outcrossing, selfing, backcrossing, locusconversion, introgression and the like.

Favorable genotypes and or marker profiles, optionally associated with atrait of interest, may be identified by one or more methodologies. Insome examples one or more markers are used, including but not limited toAFLPs, RFLPs, ASH, SSRs, SNPs, indels, padlock probes, molecularinversion probes, microarrays, sequencing, and the like. In somemethods, a target nucleic acid is amplified prior to hybridization witha probe. In other cases, the target nucleic acid is not amplified priorto hybridization, such as methods using molecular inversion probes (see,for example Hardenbol et al. (2003) Nat Biotech 21:673-678). In someexamples, the genotype related to a specific trait is monitored, whilein other examples, a genome-wide evaluation including but not limited toone or more of marker panels, library screens, association studies,microarrays, gene chips, expression studies, or sequencing such aswhole-genome resequencing and genotyping-by-sequencing (GBS) may beused. In some examples, no target-specific probe is needed, for exampleby using sequencing technologies, including but not limited tonext-generation sequencing methods (see, for example, Metzker (2010) NatRev Genet 11:31-46; and, Egan et al. (2012) Am J Bot 99:175-185) such assequencing by synthesis (e.g., Roche 454 pyrosequencing, Illumina GenomeAnalyzer, and Ion Torrent PGM or Proton systems), sequencing by ligation(e.g., SOLiD from Applied Biosystems, and Polnator system from AzcoBiotech), and single molecule sequencing (SMS or third-generationsequencing) which eliminate template amplification (e.g., Helicossystem, and PacBio RS system from Pacific BioSciences). Furthertechnologies include optical sequencing systems (e.g., Starlight fromLife Technologies), and nanopore sequencing (e.g., GridION from OxfordNanopore Technologies). Each of these may be coupled with one or moreenrichment strategies for organellar or nuclear genomes in order toreduce the complexity of the genome under investigation via PCR,hybridization, restriction enzyme (see, e.g., Elshire et al. (2011) PLoSONE 6:e19379), and expression methods. In some examples, no referencegenome sequence is needed in order to complete the analysis. X00N540 andits plant parts can be identified through a molecular marker profile.Such plant parts may be either diploid or haploid. The plant partincludes at least one cell of the plant from which it was obtained, suchas a diploid cell, a haploid cell or a somatic cell. Also provided areplants and plant parts substantially benefiting from the use of varietyX00N540 in their development, such as variety X00N540 comprising a locusconversion.

Comparisons of Pioneer Maize Variety Hybrid X00N540

A breeder uses various methods to help determine which plants should beselected from segregating populations and ultimately which inbredvarieties will be used to develop hybrids for commercialization. Inaddition to knowledge of the germplasm and plant genetics, a part of thehybrid selection process is dependent on experimental design coupledwith the use of statistical analysis. Experimental design andstatistical analysis are used to help determine which hybridcombinations are significantly better or different for one or moretraits of interest. Experimental design methods are used to assess errorso that differences between two hybrid varieties can be more accuratelyevaluated. Statistical analysis includes the calculation of mean values,determination of the statistical significance of the sources ofvariation, and the calculation of the appropriate variance components.One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see Fehr, Walt, Principles of CultivarDevelopment, pp. 261-286 (1987). Mean trait values may be used todetermine whether trait differences are significant. Trait values shouldpreferably be measured on plants grown under the same environmentalconditions, and environmental conditions should be appropriate for thetraits or traits being evaluated. Sufficient selection pressure shouldbe present for optimum measurement of traits of interest such asherbicide tolerance or herbicide, insect or disease resistance. Forexample, a locus conversion of X00N540 for herbicide resistance ortolerance should be compared with an isogenic counterpart in the absenceof the herbicide. In addition, a locus conversion for insect or diseaseresistance should be compared to the isogenic counterpart, in theabsence of disease pressure or insect pressure.

BLUP, Best Linear Unbiased Prediction, values can be reported for maizehybrid X00N540 and/or maize hybrid X00N540 comprising locus conversions.BLUP values can also be reported for other hybrids adapted to the samegrowing region as maize hybrid X00N540 with corresponding locusconversions.

Development of Maize Hybrids using X00N540

During the inbreeding process in maize, the vigor of the varietiesdecreases. However, vigor is restored when two different inbredvarieties are crossed to produce the hybrid progeny (F1). An importantconsequence of the homozygosity and homogeneity of the inbred varietiesis that the hybrid between a defined pair of inbreds may be reproducedindefinitely as long as the homogeneity of the inbred parents ismaintained. Once the inbreds that create a superior hybrid have beenidentified, a continual supply of the hybrid seed can be produced usingthese inbred parents and the hybrid corn plants can then be generatedfrom this hybrid seed supply.

X00N540 may also be used to produce a double cross hybrid or a three-wayhybrid. A single cross hybrid is produced when two inbred varieties arecrossed to produce the F1 progeny. A double cross hybrid is producedfrom four inbred varieties crossed in pairs (A×B and C×D) and then thetwo F1 hybrids are crossed again (A×B)×(C×D). A three-way cross hybridis produced from three inbred varieties where two of the inbredvarieties are crossed (A×B) and then the resulting F1 hybrid is crossedwith the third inbred variety (A×B)×C. In each case, pericarp tissuefrom the female parent will be a part of and protect the hybrid seed.

Another form of commercial hybrid production involves the use of amixture of male sterile hybrid seed and male pollinator seed. Whenplanted, the resulting male sterile hybrid plants are pollinated by thepollinator plants. This method can be used to produce grain withenhanced quality grain traits, such as high oil, because desired qualitygrain traits expressed in the pollinator will also be expressed in thegrain produced on the male sterile hybrid plant. In this method thedesired quality grain trait does not have to be incorporated by lengthyprocedures such as recurrent backcross selection into an inbred parentline. One use of this method is described in U.S. Pat. Nos. 5,704,160and 5,706,603.

Molecular data from X00N540 may be used in a plant breeding process.Nucleic acids may be isolated from a seed of X00N540 or from a plant,plant part, or cell produced by growing a seed of X00N540, or from aseed of X00N540 with a locus conversion, or from a plant, plant part, orcell of X00N540 with a locus conversion. One or more polymorphisms maybe isolated from the nucleic acids. A plant having one or more of theidentified polymorphisms may be selected and used in a plant breedingmethod to produce another plant.

Introduction of a New Trait or Locus into Hybrid Maize Variety X00N540

Hybrid variety X00N540 represents a new base genetic line into which anew locus or trait may be introduced or introgressed. Transformation andbackcrossing represent two methods that can be used to accomplish suchan introgression. The term locus conversion is used to designate theproduct of such an introgression.

To select and develop a superior hybrid, it is necessary to identify andselect genetically unique individuals that occur in a segregatingpopulation. The segregating population is the result of a combination ofcrossover events plus the independent assortment of specificcombinations of alleles at many gene loci that results in specific andunique genotypes. Once such a variety is developed its value to societyis substantial since it is important to advance the germplasm base as awhole in order to maintain or improve traits such as yield, diseaseresistance, pest resistance and plant performance in extreme weatherconditions. Locus conversions are routinely used to add or modify one ora few traits of such a line and this further enhances its value andusefulness to society.

Backcrossing can be used to improve inbred varieties and a hybridvariety which is made using those inbreds. Backcrossing can be used totransfer a specific desirable trait from one variety, the donor parent,to an inbred called the recurrent parent which has overall goodagronomic characteristics yet that lacks the desirable trait. Thistransfer of the desirable trait into an inbred with overall goodagronomic characteristics can be accomplished by first crossing arecurrent parent to a donor parent (non-recurrent parent). The progenyof this cross is then mated back to the recurrent parent followed byselection in the resultant progeny for the desired trait to betransferred from the non-recurrent parent.

Traits may be used by those of ordinary skill in the art to characterizeprogeny. Traits are commonly evaluated at a significance level, such asa 1%, 5% or 10% significance level, when measured in plants grown in thesame environmental conditions. For example, a locus conversion ofX00N540 may be characterized as having essentially the same oressentially all of the phenotypic traits or physiological andmorphological traits or characteristics as X00N540. By essentially allof the phenotypic characteristics or morphological and physiologicalcharacteristics, it is meant that all of the characteristics of a plantare recovered that are otherwise present when compared in the sameenvironment, other than an occasional variant trait that might ariseduring backcrossing or direct introduction of a transgene or geneticmodification. The traits used for comparison may be those traits shownin Table 1 as determined at the 5% significance level when grown underthe same environmental conditions. Molecular markers can also be usedduring the breeding process for the selection of qualitative traits. Forexample, markers can be used to select plants that contain the allelesof interest during a backcrossing breeding program. The markers can alsobe used to select for the genome of the recurrent parent and against thegenome of the donor parent. Using this procedure can minimize the amountof genome from the donor parent that remains in the selected plants.

A backcross or locus conversion of X00N540 can be developed when DNAsequences are introduced through backcrossing (Hallauer et al., in Cornand Corn Improvement, Sprague and Dudley, Third Ed. 1998), with a parentof X00N540 utilized as the recurrent parent. Naturally occurring,modified and transgenic DNA sequences may be introduced throughbackcrossing techniques. A backcross or locus conversion may produce aplant with a trait or locus conversion in at least one or morebackcrosses, including at least 2 backcrosses, at least 3 backcrosses,at least 4 backcrosses, at least 5 backcrosses and the like. Molecularmarker assisted breeding or selection may be utilized to reduce thenumber of backcrosses necessary to achieve the backcross conversion. Forexample, see Openshaw, et aL, “Marker-assisted Selection in BackcrossBreeding” in: Proceedings Symposium of the Analysis of Molecular Data,August 1994, Crop Science Society of America, Corvallis, Oreg., whichdemonstrated that a backcross locus conversion can be made in as few astwo backcrosses.

The complexity of the backcross conversion method depends on the type oftrait being transferred (a single gene or closely linked genes comparedto unlinked genes), the level of expression of the trait, the type ofinheritance (cytoplasmic or nuclear), dominant or recessive traitexpression, and the types of parents included in the cross. It isunderstood by those of ordinary skill in the art that for single locusor gene traits that are relatively easy to classify, the backcrossmethod is effective and relatively easy to manage. (See Hallauer et al.in Corn and Corn Improvement, Sprague and Dudley, Third Ed. 1998).Desired traits that may be transferred through backcross conversioninclude, but are not limited to, waxy starch, sterility (nuclear andcytoplasmic), fertility restoration, grain color (white), nutritionalenhancements, drought tolerance, nitrogen utilization, altered fattyacid profile, increased digestibility, low phytate, industrialenhancements, disease resistance (bacterial, fungal, or viral), insectresistance, and herbicide tolerance or resistance. A locus conversion,also called a trait conversion, can be a native trait or a transgenictrait. In addition, a recombination site itself, such as an FRT site,Lox site or other site specific integration site, may be inserted bybackcrossing and utilized for direct insertion of one or more genes ofinterest into a specific plant variety. The trait of interest istransferred from the donor parent to the recurrent parent, in this case,an inbred parent of the maize variety disclosed herein.

A single locus may contain several transgenes, such as a transgene fordisease resistance that, in the same expression vector, also contains atransgene for herbicide tolerance or resistance. The gene for herbicidetolerance or resistance may be used as a selectable marker and/or as aphenotypic trait. A single locus conversion of a site specificintegration system allows for the integration of multiple genes at aknown recombination site in the genome. At least one, at least two or atleast three and less than ten, less than nine, less than eight, lessthan seven, less than six, less than five or less than four locusconversions may be introduced into the plant by backcrossing,introgression or transformation to express the desired trait, while theplant, or a plant grown from the seed, plant part or plant cell,otherwise retains the phenotypic characteristics of the deposited seedwhen grown under the same environmental conditions.

The backcross or locus conversion may result from either the transfer ofa dominant allele or a recessive allele. Selection of progeny containingthe trait of interest can be accomplished by direct selection for atrait associated with a dominant allele. Transgenes transferred viabackcrossing typically function as a dominant single gene trait and arerelatively easy to classify. Selection of progeny for a trait that istransferred via a recessive allele, such as the waxy starchcharacteristic, requires growing and selfing the first backcrossgeneration to determine which plants carry the recessive alleles.Recessive traits may require additional progeny testing in successivebackcross generations to determine the presence of the locus ofinterest. The last backcross generation is usually selfed to give purebreeding progeny for the gene(s) being transferred, although a backcrossconversion with a stably introgressed trait may also be maintained byfurther backcrossing to the recurrent parent with selection for theconverted trait.

Along with selection for the trait of interest, progeny are selected forthe phenotype and/or genotype of the recurrent parent. Whileoccasionally additional polynucleotide sequences or genes may betransferred along with the backcross conversion, the backcrossconversion variety “fits into the same hybrid combination as therecurrent parent inbred variety and contributes the effect of theadditional locus added through the backcross.” See Poehlman et al.(1995) Breeding Field Crop, 4th Ed., Iowa State University Press, Ames,Iowa, pp. 132-155 and 321-344.

When one or more traits are introgressed into the variety a differencein quantitative agronomic traits, such as yield or dry down, between thevariety and an introgressed version of the variety in some environmentsmay occur. For example, the introgressed version, may provide a netyield increase in environments where the trait provides a benefit, suchas when a variety with an introgressed trait for insect resistance isgrown in an environment where insect pressure exists, or when a varietywith herbicide tolerance is grown in an environment where the herbicideis used.

The modified X00N540 may be further characterized as having essentiallythe same phenotypic characteristics of maize variety X00N540 such as arelisted in Table 1 when grown under the same or similar environmentalconditions and/or may be characterized by percent identity to X00N540 asdetermined by molecular markers, such as SSR markers or SNP markers.Examples of percent identity determined using markers include at least95%, 96%, 97%, 98%, 99% or 99.5%.

Traits can be used by those of ordinary skill in the art to characterizeprogeny. Traits are commonly evaluated at a significance level, such asa 1%, 5% or 10% significance level, when measured in plants grown in thesame environmental conditions.

Male Sterility and Hybrid Seed Production

Hybrid seed production requires elimination or inactivation of pollenproduced by the female inbred parent. Incomplete removal or inactivationof the pollen provides the potential for self-pollination. A reliablemethod of controlling male fertility in plants offers the opportunityfor improved seed production. There are several ways in which a maizeplant can be manipulated so that it is male sterile. These include useof manual or mechanical emasculation (or detasseling), use of one ormore genetic factors that confer male sterility, including cytoplasmicgenetic and/or nuclear genetic male sterility, use of gametocides andthe like. A male sterile variety designated X00N540 may include one ormore genetic factors, which result in cytoplasmic genetic and/or nucleargenetic male sterility. The male sterility may be either partial orcomplete male sterility.

Hybrid maize seed is often produced by a male sterility systemincorporating manual or mechanical detasseling. Alternate strips of twoinbred varieties of maize are planted in a field, and the pollen-bearingtassels are removed from one of the inbreds (female). Provided thatthere is sufficient isolation from sources of foreign maize pollen, theears of the detasseled inbred will be fertilized only from the otherinbred (male), and the resulting seed is therefore hybrid and will formhybrid plants.

Large scale commercial maize hybrid production, as it is practicedtoday, requires the use of some form of male sterility system whichcontrols or inactivates male fertility. A reliable method of controllingmale fertility in plants also offers the opportunity for improved plantbreeding. This is especially true for development of maize hybrids,which relies upon some sort of male sterility system. There are severalways in which a maize plant can be manipulated so that is male sterile.These include use of manual or mechanical emasculation (or detasseling),cytoplasmic genetic male sterility, nuclear genetic male sterility,gametocides and the like.

The laborious detasseling process can be avoided by using cytoplasmicmale-sterile (CMS) inbreds. Plants of a CMS inbred are male sterile as aresult of genetic factors in the cytoplasm, as opposed to the nucleus,and so nuclear linked genes are not transferred during backcrossing.Thus, this characteristic is inherited exclusively through the femaleparent in maize plants, since only the female provides cytoplasm to thefertilized seed. CMS plants are fertilized with pollen from anotherinbred that is not male-sterile. Pollen from the second inbred may ormay not contribute genes that make the hybrid plants male-fertile, andeither option may be preferred depending on the intended use of thehybrid. The same hybrid seed, a portion produced from detasseled fertilemaize and a portion produced using the CMS system can be blended toinsure that adequate pollen loads are available for fertilization whenthe hybrid plants are grown. CMS systems have been successfully usedsince the 1950's, and the male sterility trait is routinely backcrossedinto inbred varieties. See Wych, Robert D. (1988) “Production of HybridSeed”, Corn and Corn Improvement, Ch. 9, pp. 565-607.

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar et al. and chromosomal translocations asdescribed by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Inaddition to these methods, Albertsen et al., U.S. Pat. No. 5,432,068,describes a system of nuclear male sterility which includes: identifyinga gene which is needed for male fertility; silencing this native genewhich is needed for male fertility; removing the native promoter fromthe essential male fertility gene and replacing it with an induciblepromoter; inserting this genetically engineered gene back into theplant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing, or turning “on”,the promoter, which in turn allows the gene that confers male fertilityto be transcribed.

These, and the other methods of conferring genetic male sterility in theart, each possess their own benefits and drawbacks. Some other methodsuse a variety of approaches such as delivering into the plant a geneencoding a cytotoxic substance associated with a male tissue specificpromoter or an antisense system in which a gene needed for fertility isidentified and an antisense to that gene is inserted in the plant (seeFabinjanski, et al. EPO 89/3010153.8 publication no. 329,308 and PCTapplication PCT/CA90/00037 published as WO 90/08828).

Another system for controlling male sterility makes use of gametocides.Gametocides are not a genetic system, but rather a topical applicationof chemicals. These chemicals affect cells that are needed for malefertility. The application of these chemicals affects fertility in theplants only for the growing season in which the gametocide is applied(see Carlson, Glenn R., and U.S. Pat. No. 4,936,904). Application of thegametocide, timing of the application and genotype specificity oftenlimit the usefulness of the approach and it is not appropriate in allsituations.

Transformation

Transgenes and transformation methods facilitate engineering of thegenome of plants to contain and express heterologous genetic elements,such as foreign genetic elements, or additional copies of endogenouselements, or modified versions of native or endogenous genetic elementsin order to alter at least one trait of a plant in a specific manner.Any sequences, such as DNA, whether from a different species or from thesame species, which have been stably inserted into a genome usingtransformation are referred to herein collectively as “transgenes”and/or “transgenic events”. Transgenes can be moved from one genome toanother using breeding techniques which may include, for example,crossing, backcrossing or double haploid production. In someembodiments, a transformed variant of X00N540 may comprise at least onetransgene but could contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10and/or no more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2.Transformed versions of the claimed maize variety X00N540 containing andinheriting the transgene thereof are provided.

Numerous methods for plant transformation have been developed, includingbiological and physical plant transformation protocols. In addition,expression vectors and in vitro culture methods for plant cell or tissuetransformation and regeneration of plants are available. See, forexample, Armstrong, “The First Decade of Maize Transformation: A Reviewand Future Perspective” (Maydica 44:101-109, 1999) and Qiudeng, Q. etal. (2014) Maize transformation technology development for commercialevent generation, Frontiers in Plant Science 5: 379.

In general, methods to transform, modify, edit or alter plant endogenousgenomic DNA include altering the plant native DNA sequence or apre-existing transgenic sequence including regulatory elements, codingand non-coding sequences. These methods can be used, for example, totarget nucleic acids to pre-engineered target recognition sequences inthe genome. Such pre-engineered target sequences may be introduced bygenome editing or modification. As an example, a genetically modifiedplant variety is generated using “custom” or engineered endonucleasessuch as meganucleases produced to modify plant genomes (see e.g., WO2009/114321; Gao et al. (2010) Plant Journal 1:176-187). Anothersite-directed engineering method is through the use of zinc fingerdomain recognition coupled with the restriction properties ofrestriction enzyme. See e.g., Urnov, et al., (2010) Nat Rev Genet.11(9):636-46; Shukla, et al., (2009) Nature 459 (7245):437-41. Atranscription activator-like (TAL) effector-DNA modifying enzyme (TALEor TALEN) is also used to engineer changes in plant genome. See e.g.,US20110145940, Cermak et al., (2011) Nucleic Acids Res. 39(12) and Bochet al., (2009), Science 326(5959): 1509-12. Site-specific modificationof plant genomes can also be performed using the bacterial type IICRISPR (clustered regularly interspaced short palindromic repeats)/Cas(CRISPR-associated) system. See e.g., Belhaj et al., (2013), PlantMethods 9: 39; The Cas9/guide RNA-based system allows targeted cleavageof genomic DNA guided by a customizable small noncoding RNA in plants(see e.g., WO 2015026883A1).

Plant transformation methods may involve the construction of anexpression vector. Such a vector comprises a DNA sequence that containsa gene under the control of or operatively linked to a regulatoryelement, for example a promoter. The vector may contain one or moregenes and one or more regulatory elements.

A transgenic event which has been stably engineered into the germ cellline of a particular maize plant using transformation techniques, couldbe moved into the germ cell line of another variety using traditionalbreeding techniques that are well known in the plant breeding arts.These varieties can then be crossed to generate a hybrid maize varietyplant such as maize variety plant X00N540 which comprises a transgenicevent. For example, a backcrossing approach is commonly used to move atransgenic event from a transformed maize plant to another variety, andthe resulting progeny would then comprise the transgenic event(s). Also,if an inbred variety was used for the transformation then the transgenicplants could be crossed to a different inbred in order to produce atransgenic hybrid maize plant.

Various genetic elements can be introduced into the plant genome usingtransformation. These elements include, but are not limited to genes;coding sequences; inducible, constitutive, and tissue specificpromoters; enhancing sequences; and signal and targeting sequences. Forexample, see the traits, genes and transformation methods listed in U.S.Pat. Nos. 6,118,055 and 6,284,953. In addition, transformability of avariety can be increased by introgressing the trait of hightransformability from another variety known to have hightransformability, such as Hi-II. See U.S. Patent Application PublicationUS 2004/0016030 (2004).

With transgenic or genetically modified plants, a foreign protein can beproduced in commercial quantities. Thus, techniques for the selectionand propagation of transformed plants, which are well understood in theart, yield a plurality of transgenic or genetically modified plants thatare harvested in a conventional manner, and a foreign protein then canbe extracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Sack, M. et al., Curr. Opin. Biotech 32:163-170 (2015).

Transgenic events can be mapped by one of ordinary skill in the art andsuch techniques are well known to those of ordinary skill in the art.For exemplary methodologies in this regard, see for example, Glick andThompson, Methods in Plant Molecular Biology and Biotechnology, 269-284(CRC Press, Boca Raton, 1993).

Plants can be genetically engineered or modified to express variousphenotypes of agronomic interest. Through the transformation ormodification of maize the expression of genes can be altered to enhancedisease resistance, insect resistance, herbicide tolerance, agronomictraits, grain quality and other traits. Transformation can also be usedto insert DNA sequences which control or help control male-sterility.DNA sequences native to maize as well as non-native DNA sequences can betransformed into maize and used to alter levels of native or non-nativeproteins. Various promoters, targeting sequences, enhancing sequences,and other DNA sequences can be inserted into the maize genome for thepurpose of altering the expression of proteins. Reduction of theactivity of specific genes (also known as gene silencing, or genesuppression) is desirable for several aspects of genetic engineering inplants.

Many techniques for gene silencing are well known to one of skill in theart, including but not limited to knock-outs (such as by insertion of atransposable element such as mu (Vicki Chandler, The Maize Handbook Ch.118 (Springer-Verlag 1994) or other genetic elements such as a FRT, Loxor other site specific integration site, antisense technology (see,e.g., U.S. Pat. Nos. 5,107,065; 5,453,566; and 5,759,829);co-suppression (e.g., Taylor (1997) Plant Cell 9:1245; RNA interference(U.S. Pat. No. 5,034,323; Sharp (1999) Genes Dev. 13:139-141; Zamore etal. (2000) Cell 101:25-33; and Montgomery et al. (1998) PNAS USA95:15502-15507), virus-induced gene silencing (Burton, et al. (2000)Plant Cell 12:691-705; and Baulcombe (1999) Curr. Op. Plant Bio.2:109-113); target-RNA-specific ribozymes (Haseloff et al. (1988) Nature334: 585-591); hairpin structures (Smith et al. (2000) Nature407:319-320; WO 99/53050; and WO 98/53083); MicroRNA (Aukerman and Sakai(2003) Plant Cell 15:2730-2741); ribozymes (Steinecke et al. (1992) EMBOJ. 11:1525; and Perriman et al. (1993) Antisense Res. Dev. 3:253);oligonucleotide mediated targeted modification (e.g., WO 03/076574 andWO 99/25853); Zn-finger targeted molecules (e.g., WO 01/52620; WO03/048345; and WO 00/42219); and other methods or combinations of theabove methods known to those of skill in the art.

Exemplary nucleotide sequences that may be altered by geneticengineering include, but are not limited to, those categorized below.

1. Transgenes that Confer Resistance to Insects or Disease and thatEncode:

(A) Plant disease resistance genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266: 789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262: 1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinoset al., Cell 78: 1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae), McDowell and Woffenden, (2003) Trends Biotechnol.21(4): 178-83 and Toyoda et al., (2002) Transgenic Res. 11(6):567-82. Aplant resistant to a disease is one that is more resistant to a pathogenas compared to the wild type plant.

(B) A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon. See, for example, Geiser et al.,Gene 48: 109 (1986), who disclose the cloning and nucleotide sequence ofa Bt delta-endotoxin gene. Moreover, DNA molecules encodingdelta-endotoxin genes can be purchased from American Type CultureCollection (Rockville, Md.), for example, under ATCC Accession Nos.40098, 67136, 31995 and 31998. Other non-limiting examples of Bacillusthuringiensis transgenes being genetically engineered are given in thefollowing patents and patent applications: U.S. Pat. Nos. 5,188,960;5,689,052; 5,880,275; 5,986,177; 7,105,332; 7,208,474; WO 91/14778; WO99/31248; WO 01/12731; WO 99/24581; WO 97/40162 and U.S. applicationSer. Nos. 10/032,717; 10/414,637; 11/018,615; 11/404,297; 11/404,638;11/471,878; 11/780,501; 11/780,511; 11/780,503; 11/953,648; and Ser. No.11/957,893.

(C) An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344: 458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

(D) An insect-specific peptide which, upon expression, disrupts thephysiology of the affected pest. For example, see the disclosures ofRegan, J. Biol. Chem. 269: 9 (1994) (expression cloning yields DNAcoding for insect diuretic hormone receptor); Pratt et al., Biochem.Biophys. Res. Comm. 163: 1243 (1989) (an allostatin is identified inDiploptera puntata); Chattopadhyay et al. (2004) Critical Reviews inMicrobiology 30 (1): 33-54 2004; Zjawiony (2004) J Nat Prod 67 (2):300-310; Carlini and Grossi-de-Sa (2002) Toxicon, 40 (11): 1515-1539;Ussuf et al. (2001) Curr Sci. 80 (7): 847-853; and Vasconcelos andOliveira (2004) Toxicon 44 (4): 385-403. See also U.S. Pat. No.5,266,317 to Tomalski et al., who disclose genes encodinginsect-specific toxins.

(E) An enzyme responsible for a hyperaccumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

(F) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23: 691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21: 673 (1993), who provide the nucleotide sequenceof the parsley ubi4-2 polyubiquitin gene, and U.S. Pat. Nos. 6,563,020;7,145,060 and 7,087,810.

(G) A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24: 757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104: 1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

(H) A hydrophobic moment peptide. See PCT application WO 95/16776 andU.S. Pat. No. 5,580,852 disclosure of peptide derivatives of Tachyplesinwhich inhibit fungal plant pathogens) and PCT application WO 95/18855and U.S. Pat. No. 5,607,914 (teaches synthetic antimicrobial peptidesthat confer disease resistance).

(I) A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure by Jaynes et al., Plant Sci. 89: 43 (1993),of heterologous expression of a cecropin-beta lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

(J) A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28: 451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

(K) An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, SEVENTH INT'L SYMPOSIUM ON MOLECULARPLANT-MICROBE INTERACTIONS (Edinburgh, Scotland, 1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

(L) A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366: 469 (1993), which shows that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

(M) A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo alpha-1,4-D-polygalacturonasesfacilitate fungal colonization and plant nutrient release bysolubilizing plant cell wall homo-alpha-1,4-D-galacturonase. See Lamb etal., Bio/Technology 10: 1436 (1992). The cloning and characterization ofa gene which encodes a bean endopolygalacturonase-inhibiting protein isdescribed by Toubart et al., Plant J. 2: 367 (1992).

(N) A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10: 305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

(O) Genes involved in the Systemic Acquired Resistance (SAR) Responseand/or the pathogenesis related genes. Briggs, S., Current Biology, 5(2)(1995), Pieterse and Van Loon (2004) Curr. Opin. Plant Bio. 7(4):456-64and Somssich (2003) Cell 113(7):815-6.

(P) Antifungal genes (Cornelissen and Melchers, Pl. Physiol.101:709-712, (1993) and Parijs et al., Planta 183:258-264, (1991) andBushnell et al., Can. J. of Plant Path. 20(2):137-149 (1998). Also seeU.S. application Ser. Nos. 09/950,933; 11/619,645; 11/657,710;11/748,994; 11/774,121 and U.S. Pat. Nos. 6,891,085 and 7,306,946.

(Q) Detoxification genes, such as for fumonisin, beauvericin,moniliformin and zearalenone and their structurally related derivatives.For example, see U.S. Pat. Nos. 5,716,820; 5,792,931; 5,798,255;5,846,812; 6,083,736; 6,538,177; 6,388,171 and 6,812,380.

(R) Cystatin and cysteine proteinase inhibitors. See U.S. Pat. No.7,205,453.

(S) Defensin genes. See WO03000863 and U.S. Pat. Nos. 6,911,577;6,855,865; 6,777,592 and 7,238,781.

(T) Genes conferring resistance to nematodes. See e.g. PCT ApplicationWO96/30517; PCT Application WO93/19181, WO 03/033651 and Urwin et al.,Planta 204:472-479 (1998), Williamson (1999) Curr Opin Plant Bio.2(4):327-31; and U.S. Pat. Nos. 6,284,948 and 7,301,069.

-   -   (U) Genes that confer resistance to Phytophthora Root Rot, such        as the Rps 1, Rps 1-a, Rps 1-b, Rps 1-c, Rps 1-d, Rps 1-e, Rps        1-k, Rps 2, Rps 3-a, Rps 3-b, Rps 3-c, Rps 4, Rps 5, Rps 6, Rps        7 and other Rps genes. See, for example, Shoemaker et al,        Phytophthora Root Rot Resistance Gene Mapping in Soybean, Plant        Genome IV Conference, San Diego, Calif. (1995).

(V) Genes that confer resistance to Brown Stem Rot, such as described inU.S. Pat. No. 5,689,035.

(W) Genes that confer resistance to Colletotrichum, such as described inUS Patent publication US20090035765. This includes the Rcg locus thatmay be utilized as a single locus conversion.

2. Transgenes that Confer Tolerance to a Herbicide, for Example:

(A) A herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant acetolactate synthase (ALS) and acetohydroxyacid synthase(AHAS) enzyme as described, for example, in U.S. Pat. Nos. 5,605,011;5,013,659; 5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373;5,331,107; 5,928,937; and 5,378,824; US Patent Publication No.20070214515, and international publication WO 96/33270.

(B) Glyphosate (tolerance imparted by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus phosphinothricin acetyl transferase (bar) genes), andpyridinoxy or phenoxy proprionic acids and cyclohexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835,which discloses the nucleotide sequence of a form of EPSPS which canconfer glyphosate tolerance. U.S. Pat. No. 5,627,061 also describesgenes encoding EPSPS enzymes. See also U.S. Pat. Nos. 6,566,587;6,338,961; 6,248,876 B1; 6,040,497; 5,804,425; 5,633,435; 5,145,783;4,971,908; 5,312,910; 5,188,642; 4,940,835; 5,866,775; 6,225,114 B1;6,130,366; 5,310,667; 4,535,060; 4,769,061; 5,633,448; 5,510,471; Re.36,449; RE 37,287 E; and 5,491,288; and international publicationsEP1173580; WO 01/66704; EP1173581 and EP1173582.

Glyphosate tolerance is also imparted to plants that express a gene thatencodes a glyphosate oxido-reductase enzyme as described more fully inU.S. Pat. Nos. 5,776,760 and 5,463,175. In addition, glyphosatetolerance can be imparted to plants by the over expression of genesencoding glyphosate N-acetyltransferase. See, for example,US2004/0082770; US2005/0246798; and US2008/0234130. A DNA moleculeencoding a mutant aroA gene can be obtained under ATCC accession No.39256, and the nucleotide sequence of the mutant gene is disclosed inU.S. Pat. No. 4,769,061. European Patent Application No. 0 333 033 andU.S. Pat. No. 4,975,374 disclose nucleotide sequences of glutaminesynthetase genes which confer tolerance to herbicides such asL-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in European PatentNos. 0 242 246 and 0 242 236. See also, U.S. Pat. Nos. 5,969,213;5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477;5,646,024; 6,177,616 B1; and 5,879,903. Exemplary genes conferringresistance to phenoxy propionic acids, cyclohexanediones andcyclohexones, such as sethoxydim and haloxyfop, are the Acc1-S1, Acc1-S2and Acc1-S3 genes described by Marshall et al., Theor. Appl. Genet. 83:435 (1992).

(C) A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene) such as bromoxynil.Przibilla et al., Plant Cell 3: 169 (1991), describe the transformationof Chlamydomonas with plasmids encoding mutant psbA genes. Nucleotidesequences for nitrilase genes are disclosed in U.S. Pat. No. 4,810,648to Stalker, and DNA molecules containing these genes are available underATCC Accession Nos. 53435, 67441 and 67442. Cloning and expression ofDNA coding for a glutathione S-transferase is described by Hayes et al.,Biochem. J. 285: 173 (1992).

(D) Other genes that confer tolerance to herbicides include: a geneencoding a chimeric protein of rat cytochrome P4507A1 and yeastNADPH-cytochrome P450 oxidoreductase (Shiota et al. (1994) Plant Physiol106:17), genes for glutathione reductase and superoxide dismutase (Aonoet al. (1995) Plant Cell Physiol 36:1687, and genes for variousphosphotransferases (Datta et al. (1992) Plant Mol Biol 20:619).

(E) A herbicide that inhibits protoporphyrinogen oxidase (protox or PPO)is necessary for the production of chlorophyll, which is necessary forall plant survival. The protox enzyme serves as the target for a varietyof herbicidal compounds. PPO-inbibitor herbicides can inhibit growth ofall the different species of plants present, causing their totaldestruction. The development of plants containing altered protoxactivity which are tolerant to these herbicides are described, forexample, in U.S. Pat. Nos. 6,288,306 B1; 6,282,837 B1; and 5,767,373;and international patent publication WO 01/12825.

(F) Dicamba (3,6-dichloro-2-methoxybenzoic acid) is an organochloridederivative of benzoic acid which functions by increasing plant growthrate such that the plant dies.

3. Transgenes that Confer or Contribute to an Altered GrainCharacteristic, Such as:

(A) Altered fatty acids, for example, by

-   -   (1) Down-regulation of stearoyl-ACP desaturase to increase        stearic acid content of the plant. See Knultzon et al., Proc.        Natl. Acad. Sci. USA 89: 2624 (1992) and WO99/64579 (Genes for        Desaturases to Alter Lipid Profiles in Corn),    -   (2) Elevating oleic acid via FAD-2 gene modification and/or        decreasing linolenic acid via FAD-3 gene modification (see U.S.        Pat. Nos. 6,063,947; 6,323,392; 6,372,965 and WO 93/11245),    -   (3) Altering conjugated linolenic or linoleic acid content, such        as in WO 01/12800,    -   (4) Altering LEC1, AGP, Dek1, Superal1, mi1ps, various Ipa genes        such as Ipa1, Ipa3, hpt or hggt. For example, see WO 02/42424,        WO 98/22604, WO 03/011015, WO02/057439, WO03/011015, U.S. Pat.        Nos. 6,423,886, 6,197,561, 6,825,397, and U.S. Application        Serial Nos. US2003/0079247, US2003/0204870, and        Rivera-Madrid, R. et al. Proc. Natl. Acad. Sci. 92:5620-5624        (1995).

B) Altered phosphate content, for example, by the

-   -   (1) Introduction of a phytase-encoding gene would enhance        breakdown of phytate, adding more free phosphate to the        transformed plant. For example, see Van Hartingsveldt et al.,        Gene 127: 87 (1993), for a disclosure of the nucleotide sequence        of an Aspergillus niger phytase gene.    -   (2) Modulating a gene that reduces phytate content. In maize,        this, for example, could be accomplished, by cloning and then        re-introducing DNA associated with one or more of the alleles,        such as the LPA alleles, identified in maize mutants        characterized by low levels of phytic acid, such as in WO        05/113778 and/or by altering inositol kinase activity as in WO        02/059324, US2003/0009011, WO 03/027243, US2003/0079247, WO        99/05298, U.S. Pat. Nos. 6,197,561, 6,291,224, 6,391,348,        WO2002/059324, US2003/0079247, Wo98/45448, WO99/55882,        WO01/04147.

(C) Altered carbohydrates affected, for example, by altering a gene foran enzyme that affects the branching pattern of starch or, a genealtering thioredoxin such as NTR and/or TRX (See U.S. Pat. No.6,531,648) and/or a gamma zein knock out or mutant such as cs27 orTUSC27 or en27 (See U.S. Pat. No. 6,858,778 and US2005/0160488,US2005/0204418). See Shiroza et al., J. Bacteriol. 170: 810 (1988)(nucleotide sequence of Streptococcus mutans fructosyltransferase gene),Pen et al., Bio/Technology 10: 292 (1992) (production of transgenicplants that express Bacillus licheniformis alpha-amylase), Elliot etal., Plant Molec. Biol. 21: 515 (1993) (nucleotide sequences of tomatoinvertase genes), Søgaard et al., J. Biol. Chem. 268: 22480 (1993)(site-directed mutagenesis of barley alpha-amylase gene), and Fisher etal., Plant Physiol. 102: 1045 (1993) (maize endosperm starch branchingenzyme II), WO 99/10498 (improved digestibility and/or starch extractionthrough modification of UDP-D-xylose 4-epimerase, Fragile 1 and 2, Ref1,HCHL, C4H), U.S. Pat. No. 6,232,529 (method of producing high oil seedby modification of starch levels (AGP)). The fatty acid modificationgenes mentioned herein may also be used to affect starch content and/orcomposition through the interrelationship of the starch and oilpathways.

(D) Altered antioxidant content or composition, such as alteration oftocopherol or tocotrienols. For example, see U.S. Pat. No. 6,787,683,US2004/0034886 and WO 00/68393 involving the manipulation of antioxidantlevels, and WO 03/082899 through alteration of a homogentisate geranyltransferase (hggt).

(E) Altered essential seed amino acids. For example, see U.S. Pat. No.6,127,600 (method of increasing accumulation of essential amino acids inseeds), U.S. Pat. No. 6,080,913 (binary methods of increasingaccumulation of essential amino acids in seeds), U.S. Pat. No. 5,990,389(high lysine), WO99/40209 (alteration of amino acid compositions inseeds), WO99/29882 (methods for altering amino acid content ofproteins), U.S. Pat. No. 5,850,016 (alteration of amino acidcompositions in seeds), WO98/20133 (proteins with enhanced levels ofessential amino acids), U.S. Pat. No. 5,885,802 (high methionine), U.S.Pat. No. 5,885,801 (high threonine), U.S. Pat. No. 6,664,445 (plantamino acid biosynthetic enzymes), U.S. Pat. No. 6,459,019 (increasedlysine and threonine), U.S. Pat. No. 6,441,274 (plant tryptophansynthase beta subunit), U.S. Pat. No. 6,346,403 (methionine metabolicenzymes), U.S. Pat. No. 5,939,599 (high sulfur), U.S. Pat. No. 5,912,414(increased methionine), WO98/56935 (plant amino acid biosyntheticenzymes), WO98/45458 (engineered seed protein having higher percentageof essential amino acids), WO98/42831 (increased lysine), U.S. Pat. No.5,633,436 (increasing sulfur amino acid content), U.S. Pat. No.5,559,223 (synthetic storage proteins with defined structure containingprogrammable levels of essential amino acids for improvement of thenutritional value of plants), WO96/01905 (increased threonine),WO95/15392 (increased lysine), US2003/0163838, US2003/0150014,US2004/0068767, U.S. Pat. No. 6,803,498, WO01/79516.

4. Genes that Control Male-Sterility:

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar et al. and chromosomal translocations asdescribed by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Inaddition to these methods, Albertsen et al., U.S. Pat. No. 5,432,068,describe a system of nuclear male sterility which includes: identifyinga gene which is needed for male fertility; silencing this native genewhich is needed for male fertility; removing the native promoter fromthe essential male fertility gene and replacing it with an induciblepromoter; inserting this genetically engineered gene back into theplant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing, or turning “on”,the promoter, which in turn allows the gene that confers male fertilityto be transcribed.

(A) Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN—Ac-PPT (WO 01/29237).

(B) Introduction of various stamen-specific promoters (WO 92/13956, WO92/13957).

(C) Introduction of the barnase and the barstar gene (Paul et al. PlantMol. Biol. 19:611-622, 1992).

For additional examples of nuclear male and female sterility systems andgenes, see also, U.S. Pat. Nos. 5,859,341; 6,297,426; 5,478,369;5,824,524; 5,850,014; and 6,265,640.

5. Genes that create a site for site specific DNA integration. Thisincludes the introduction of FRT sites that may be used in the FLP/FRTsystem and/or Lox sites that may be used in the Cre/Loxp system. Forexample, see Lyznik, et al., Site-Specific Recombination for GeneticEngineering in Plants, Plant Cell Rep (2003) 21:925-932 and WO 99/25821.Other systems that may be used include the Gin recombinase of phage Mu(Maeser et aL, 1991; Vicki Chandler, The Maize Handbook Ch. 118(Springer-Verlag 1994), the Pin recombinase of E. coli (Enomoto et aL,1983), and the R/RS system of the pSR1 plasmid (Araki et al., 1992).6. Genes that affect abiotic stress resistance (including but notlimited to flowering, ear and seed development, enhancement of nitrogenutilization efficiency, altered nitrogen responsiveness, droughtresistance or tolerance, cold resistance or tolerance, and saltresistance or tolerance) and increased yield under stress. For example,see: WO 00/73475 where water use efficiency is altered throughalteration of malate; U.S. Pat. Nos. 5,892,009; 5,965,705; 5,929,305;5,891,859; 6,417,428; 6,664,446; 6,706,866; 6,717,034; 6,801,104;WO2000060089; WO2001026459; WO2001035725; WO2001034726; WO2001035727;WO2001036444; WO2001036597; WO2001036598; WO2002015675; WO2002017430;WO2002077185; WO2002079403; WO2003013227; WO2003013228; WO2003014327;WO2004031349; WO2004076638; WO9809521; and WO9938977 describing genes,including CBF genes and transcription factors effective in mitigatingthe negative effects of freezing, high salinity, and drought on plants,as well as conferring other positive effects on plant phenotype;US2004/0148654 and WO01/36596 where abscisic acid is altered in plantsresulting in improved plant phenotype such as increased yield and/orincreased tolerance to abiotic stress; WO2000/006341, WO04/090143, U.S.application Ser. Nos. 10/817,483 and 09/545,334 where cytokininexpression is modified resulting in plants with increased stresstolerance, such as drought tolerance, and/or increased yield. Also seeWO0202776, WO2003052063, JP2002281975, U.S. Pat. No. 6,084,153,WO0164898, U.S. Pat. Nos. 6,177,275, and 6,107,547 (enhancement ofnitrogen utilization and altered nitrogen responsiveness). For ethylenealteration, see US20040128719, US20030166197 and WO200032761. For planttranscription factors or transcriptional regulators of abiotic stress,see e.g. US20040098764 or US20040078852.

Other genes and transcription factors that affect plant growth andagronomic traits such as yield, flowering, plant growth and/or plantstructure, can be introduced or introgressed into plants, see e.g.WO97/49811 (LHY), WO98/56918 (ESD4), WO97/10339 and U.S. Pat. No.6,573,430 (TFL), U.S. Pat. No. 6,713,663 (FT), WO96/14414 (CON),WO96/38560, WO01/21822 (VRN1), WO00/44918 (VRN2), WO99/49064 (GI),WO00/46358 (FRI), WO97/29123, U.S. Pat. Nos. 6,794,560, 6,307,126 (GAI),WO99/09174 (D8 and Rht), WO2004076638 and WO2004031349 (transcriptionfactors).

Using X00N540 to Develop Another Maize Plant

The development of maize hybrids in a maize plant breeding programrequires, in general, the development of homozygous inbred lines, thecrossing of these lines, and the evaluation of the crosses. Maize plantbreeding programs combine the genetic backgrounds from two or moreinbred varieties or various other germplasm sources into breedingpopulations from which new inbred varieties are developed by selfing andselection of desired phenotypes. Hybrids also can be used as a source ofplant breeding material or as source populations from which to developor derive new maize varieties. Plant breeding techniques known in theart and used in a maize plant breeding program include, but are notlimited to, recurrent selection, mass selection, bulk selection,backcrossing, making double haploids, pedigree breeding, openpollination breeding, restriction fragment length polymorphism enhancedselection, genetic marker enhanced selection, and transformation. Oftencombinations of these techniques are used. The inbred varieties derivedfrom hybrids can be developed using plant breeding techniques asdescribed above. New inbreds are crossed with other inbred varieties andthe hybrids from these crosses are evaluated to determine which of thosehave commercial potential. The oldest and most traditional method ofanalysis is the observation of phenotypic traits but genotypic analysismay also be used.

Methods for producing a maize plant by crossing a first parent maizeplant with a second parent maize plant wherein either the first orsecond parent maize plant is a maize plant of the variety X00N540 areprovided. The other parent may be any other maize plant, such as anotherinbred variety or a plant that is part of a synthetic or naturalpopulation. Any such methods using the maize variety X00N540 in crossingor breeding are provided, such as, for example: selfing, sibbing,backcrosses, mass selection, pedigree breeding, bulk selection, hybridproduction, crosses to populations, and the like. These methods are wellknown in the art and some of the more commonly used breeding methods aredescribed below and can be found in one of several reference books(e.g., Allard, Principles of Plant Breeding, 1960; Simmonds, Principlesof Crop Improvement, 1979; Fehr, “Breeding Methods for CultivarDevelopment”, Production and Uses, 2^(nd) ed., Wilcox editor, 1987).

Recurrent Selection and Mass Selection

Recurrent selection is a method used in a plant breeding program toimprove a population of plants. X00N540 is suitable for use in arecurrent selection program. The method entails individual plants crosspollinating with each other to form progeny. The progeny are grown andthe superior progeny selected by any number of selection methods, whichinclude individual plant, half-sib progeny, full-sib progeny, selfedprogeny and toperossing. The selected progeny are cross pollinated witheach other to form progeny for another population. This population isplanted and again superior plants are selected to cross pollinate witheach other. Recurrent selection is a cyclical process and therefore canbe repeated as many times as desired. The objective of recurrentselection is to improve the traits of a population. The improvedpopulation can then be used as a source of breeding material to obtaininbred varieties to be used in hybrids or used as parents for asynthetic cultivar. A synthetic cultivar is the resultant progeny formedby the intercrossing of several selected inbreds.

X00N540 is suitable for use in mass selection. Mass selection is auseful technique when used in conjunction with molecular marker enhancedselection. In mass selection seeds from individuals are selected basedon phenotype and/or genotype. These selected seeds are then bulked andused to grow the next generation. Bulk selection requires growing apopulation of plants in a bulk plot, allowing the plants toself-pollinate, harvesting the seed in bulk and then using a sample ofthe seed harvested in bulk to plant the next generation. Instead ofself-pollination, directed pollination could be used as part of thebreeding program.

Production of Double Haploids

The production of double haploids from X00N540 can also be used for thedevelopment of inbreds. Double haploids are produced by the doubling ofa set of chromosomes (1N) from a heterozygous plant to produce acompletely homozygous individual. For example, a method is provided ofobtaining a substantially homozygous X00N540 progeny plant by obtaininga seed from the cross of X00N540 and another maize plant and applyingdouble haploid methods to the F1 seed or F1 plant or to any successivefilial generation. Methods for producing plants by doubling haploid seedgenerated by a cross of the plants, or parts thereof, disclosed hereinwith a different maize plant are provided. The use of double haploidssubstantially decreases the number of generations required to produce aninbred with similar genetics or characteristics to X00N540. For example,see Wan et al., “Efficient Production of Doubled Haploid Plants ThroughColchicine Treatment of Anther-Derived Maize Callus”, Theoretical andApplied Genetics, 77:889-892, 1989 and U.S. Patent Application No.2003/0005479. This can be advantageous because the process omits thegenerations of selfing needed to obtain a homozygous plant from aheterozygous source.

Haploid induction systems have been developed for various plants toproduce haploid tissues, plants and seeds. The haploid induction systemcan produce haploid plants from any genotype by crossing a selectedvariety (as female) with an inducer variety. Such inducer varieties formaize include Stock 6 (Coe, 1959, Am. Nat. 93:381-382; Sharkar and Coe,1966, Genetics 54:453-464) RWS (available online from the UniversitätHohenheim), KEMS (Deimling, Roeber, and Geiger, 1997, Vortr.Pflanzenzuchtg 38:203-224), or KMS and ZMS (Chalyk, Bylich and Chebotar,1994, MNL 68:47; Chalyk and Chebotar, 2000, Plant Breeding 119:363-364),and indeterminate gametophyte (ig) mutation (Kermicle 1969 Science166:1422-1424).

Methods for obtaining haploid plants are also disclosed in, for example,Kalinowska, K., Chamas, S., Unkel, K. et al. Theor Appl Genet (2018)1-13 at citation https:// followed by doi.org/10.1007/s00122-018-3261-9;see also Eder J, Chalyk S (2002) In vivo haploid induction in maize.Theor Appl Genet 104:703-708; U.S. Pat. No. 5,639,951 and US PatentApplication Publication No. 20020188965.

In particular, a process of making seed substantially retaining themolecular marker profile of maize variety X00N540 is provided. Obtaininga seed of hybrid maize variety X00N540 further comprising a locusconversion, wherein representative seed is produced by crossing a firstplant of variety PH48RS or a locus conversion thereof with a secondplant of variety PH42V3 or a locus conversion thereof, and whereinrepresentative seed of said varieties PH48RS and PH42V3 have beendeposited and wherein said maize variety X00N540 further comprising alocus conversion has 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of thesame polymorphisms for molecular markers as the plant or plant part ofmaize variety X00N540. Sequences for the public markers can be found,for example, in the Panzea database which is available online fromPanzea. The type of molecular marker used in the molecular profile canbe but is not limited to Single Nucleotide Polymorphisms, SNPs. Aprocess of making seed retaining essentially the same phenotypic,physiological, morphological or any combination thereof characteristicsof maize variety X00N540 is also contemplated. Obtaining a seed ofhybrid maize variety X00N540 further comprising a locus conversion,wherein representative seed is produced by crossing a first plant ofvariety PH48RS or a locus conversion thereof with a second plant ofvariety PH42V3 or a locus conversion thereof, and wherein representativeseed of said varieties PH48RS and PH42V3 have been deposited and whereinsaid maize variety X00N540 further comprising a locus conversion hasessentially the same morphological characteristics as maize varietyX00N540 when grown in the same environmental conditions. The sameenvironmental conditions may be, but is not limited to, a side-by-sidecomparison. The characteristics can be or include, for example, thoselisted in Table 1. The comparison can be made using any number ofprofessionally accepted experimental designs and statistical analysis.

Use of X00N540 in Tissue Culture

Methods of tissue culturing cells of X00N540 and a tissue culture ofX00N540 is provided. As used herein, the term “tissue culture” includesplant protoplasts, plant cell tissue culture, cultured microspores,plant calli, plant clumps, and the like. In certain embodiments, thetissue culture comprises embryos, protoplasts, meristematic cells,pollen, leaves or anthers derived from immature tissues of pollen,flowers, kernels, ears, cobs, leaves, husks, stalks, roots, root tips,anthers, silk, and the like. As used herein, phrases such as “growingthe seed” or “grown from the seed” include embryo rescue, isolation ofcells from seed for use in tissue culture, as well as traditionalgrowing methods.

Means for preparing and maintaining plant tissue cultures are well knownin the art. See, e.g., U.S. Pat. Nos. 5,538,880; 5,550,318, and6,437,224, the latter describing tissue issue culture of maize,including tassel/anther culture. Thus, in certain embodiments, cells areprovided which upon growth and differentiation produce maize plantshaving the genotype and/or phenotypic characteristics of varietyX00N540.

Seed Treatments and Cleaning

Methods of harvesting the grain of the F1 plant of variety X00N540 andusing the F2 grain as seed for planting are provided. Also provided aremethods of using the seed of variety X00N540, or selfed grain harvestedfrom variety X00N540, as seed for planting. Embodiments include cleaningthe seed, treating the seed, and/or conditioning the seed and seedproduced by such cleaning, conditioning, treating or any combinationthereof. Cleaning the seed is understood in the art to include removalof one or more of foreign debris such as weed seed, chaff, and non-seedplant matter from the seed. Conditioning the seed is understood in theart to include controlling the temperature and rate of dry down of theseed and storing the seed in a controlled temperature environment. Seedtreatment is the application of a composition to the seed such as acoating or powder. Methods for producing a treated seed include the stepof applying a composition to the seed or seed surface. Seeds areprovided which have on the surface a composition. Biological activecomponents such as bacteria can also be used as a seed treatment. Someexamples of compositions include active components such as insecticides,fungicides, pesticides, antimicrobials, germination inhibitors,germination promoters, cytokinins, and nutrients. Biological activecomponents, such as bacteria, can also be used as a seed treatment.Carriers such as polymers can be used to increase binding of the activecomponent to the seed.

To protect and to enhance yield production and trait technologies, seedtreatment options can provide additional crop plan flexibility and costeffective control against insects, weeds and diseases, thereby furtherenhancing the invention described herein. Seed material can be treated,typically surface treated, with a composition comprising combinations ofchemical or biological herbicides, herbicide safeners, insecticides,fungicides, germination inhibitors and enhancers, nutrients, plantgrowth regulators and activators, bactericides, nematicides, avicidesand/or molluscicides. These compounds are typically formulated togetherwith further carriers, surfactants or application-promoting adjuvantscustomarily employed in the art of formulation. The coatings may beapplied by impregnating propagation material with a liquid formulationor by coating with a combined wet or dry formulation. Examples of thevarious types of compounds that may be used as seed treatments areprovided in The Pesticide Manual: A World Compendium, C. D. S. TomlinEd., Published by the British Crop Production Council.

Some seed treatments that may be used on crop seed include, but are notlimited to, one or more of abscisic acid, acibenzolar-S-methyl,avermectin, amitrol, azaconazole, azospirillum, azadirachtin,azoxystrobin, Bacillus spp. (including one or more of cereus, firmus,megaterium, pumilis, sphaericus, subtilis and/or thuringiensis),Bradyrhizobium spp. (including one or more of betae, canariense,elkanii, iriomotense, japonicum, liaonigense, pachyrhizi and/oryuanmingense), captan, carboxin, chitosan, clothianidin, copper,cyazypyr, difenoconazole, etidiazole, fipronil, fludioxonil,fluoxastrobin, fluquinconazole, flurazole, fluxofenim, harpin protein,imazalil, imidacloprid, ipconazole, isoflavenoids,lipo-chitooligosaccharide, mancozeb, manganese, maneb, mefenoxam,metalaxyl, metconazole, myclobutanil, PCNB, penflufen, penicillium,penthiopyrad, permethrine, picoxystrobin, prothioconazole,pyraclostrobin, rynaxypyr, S-metolachlor, saponin, sedaxane, TCMTB,tebuconazole, thiabendazole, thiamethoxam, thiocarb, thiram,tolclofos-methyl, triadimenol, trichoderma, trifloxystrobin,triticonazole and/or zinc. PCNB seed coat refers to EPA registrationnumber 00293500419, containing quintozen and terrazole. TCMTB refers to2-(thiocyanomethylthio) benzothiazole.

Seed varieties and seeds with specific transgenic traits may be testedto determine which seed treatment options and application rates maycomplement such varieties and transgenic traits in order to enhanceyield. For example, a variety with good yield potential but head smutsusceptibility may benefit from the use of a seed treatment thatprovides protection against head smut, a variety with good yieldpotential but cyst nematode susceptibility may benefit from the use of aseed treatment that provides protection against cyst nematode, and soon. Likewise, a variety encompassing a transgenic trait conferringinsect resistance may benefit from the second mode of action conferredby the seed treatment, a variety encompassing a transgenic traitconferring herbicide resistance may benefit from a seed treatment with asafener that enhances the plants resistance to that herbicide, etc.Further, the good root establishment and early emergence that resultsfrom the proper use of a seed treatment may result in more efficientnitrogen use, a better ability to withstand drought and an overallincrease in yield potential of a variety or varieties containing acertain trait when combined with a seed treatment.

INDUSTRIAL APPLICABILITY

Another embodiment is a method of harvesting the grain of the F1 plantof variety X00N540 and using the grain in a commodity. Methods ofproducing a commodity plant product are also provided. Examples of maizegrain as a commodity plant product include, but are not limited to,oils, meals, flour, starches, syrups, proteins, cellulose, silage, andsugars. Maize grain is used as human food, livestock feed, and as rawmaterial in industry. The food uses of maize, in addition to humanconsumption of maize kernels, include both products of dry- andwet-milling industries. The principal products of maize dry milling aregrits, meal and flour. The maize wet-milling industry can provide maizestarch, maize syrups, and dextrose for food use. Maize oil is recoveredfrom maize germ, which is a by-product of both dry- and wet-millingindustries. Processing the grain can include one or more of cleaning toremove foreign material and debris from the grain, conditioning, such asaddition of moisture to the grain, steeping the grain, wet milling, drymilling and sifting.

Maize, including both grain and non-grain portions of the plant, is alsoused extensively as livestock feed, primarily for beef cattle, dairycattle, hogs, and poultry.

Industrial uses of maize include production of ethanol, maize starch inthe wet-milling industry and maize flour in the dry-milling industry.The industrial applications of maize starch and flour are based onfunctional properties, such as viscosity, film formation, adhesiveproperties, and ability to suspend particles. The maize starch and flourhave application in the paper and textile industries. Other industrialuses include applications in adhesives, building materials, foundrybinders, laundry starches, explosives, oil-well muds, and other miningapplications.

Plant parts other than the grain of maize are also used in industry: forexample, stalks and husks are made into paper and wallboard and cobs areused for fuel and to make charcoal.

The seed of the maize variety, the plant produced from the seed, a plantproduced from crossing of maize variety X00N540 and various parts of themaize plant and transgenic versions of the foregoing, can be utilizedfor human food, livestock feed, and as a raw material in industry.

All publications, patents, and patent applications mentioned in thespecification are incorporated by reference herein for the purpose citedto the same extent as if each was specifically and individuallyindicated to be incorporated by reference herein.

The foregoing invention has been described in detail by way ofillustration and example for purposes of clarity and understanding. Asis readily apparent to one skilled in the art, the foregoing are onlysome of the methods and compositions that illustrate the embodiments ofthe foregoing invention. It will be apparent to those of ordinary skillin the art that variations, changes, modifications, and alterations maybe applied to the compositions and/or methods described herein withoutdeparting from the true spirit, concept, and scope of the invention.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having,” “contains”, “containing,” “characterizedby” or any other variation thereof, are intended to cover anon-exclusive inclusion.

Unless expressly stated to the contrary, “or” is used as an inclusiveterm. For example, a condition A or B is satisfied by any one of thefollowing: A is true (or present) and B is false (or not present), A isfalse (or not present) and B is true (or present), and both A and B aretrue (or present). The indefinite articles “a” and “an” preceding anelement or component are nonrestrictive regarding the number ofinstances (i.e., occurrences) of the element or component. Therefore “a”or “an” should be read to include one or at least one, and the singularword form of the element or component also includes the plural unlessthe number is obviously meant to be singular.

Deposits

Applicant has made a deposit of at least 625 seeds of parental maizeinbred varieties PH48RS and PH42V3 with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209USA, with ATCC Deposit Nos. PTA-126645 and PTA-124764, respectively. Theseeds deposited with the ATCC on Feb. 12, 2020 for PTA-126645 and onFeb. 20, 2018 for PTA-124764, were obtained from the seed of the varietymaintained by Pioneer Hi-Bred International, Inc., 7250 NW 62^(nd)Avenue, Johnston, Iowa 50131-1000 since prior to the filing date of thisapplication. Access to this seed will be available during the pendencyof the application to the Commissioner of Patents and Trademarks andpersons determined by the Commissioner to be entitled thereto uponrequest. Upon issuance of any claims in the application, the Applicantwill make available to the public, pursuant to 37 C.F.R. § 1.808, asample(s) of the deposit of at least 625 seeds of parental maize inbredvarieties PH48RS and PH42V3 with the American Type Culture Collection(ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209. Thedeposits of the seed of parental maize inbred varieties for Hybrid MaizeVariety X00N540 will be maintained in the ATCC depository, which is apublic depository, for a period of 30 years, or 5 years after the mostrecent request, or for the enforceable life of the patent, whichever islonger, and will be replaced if it becomes nonviable during that period.Additionally, Applicant has or will satisfy all of the requirements of37 C.F.R. §§ 1.801-1.809, including providing an indication of theviability of the sample upon deposit. Applicant has no authority towaive any restrictions imposed by law on the transfer of biologicalmaterial or its transportation in commerce. Applicant does not waive anyinfringement of the rights granted under this patent or rightsapplicable to Hybrid Maize Variety X00N540 and/or its parental maizeinbred varieties PH48RS and PH42V3 under either the patent laws or thePlant Variety Protection Act (7 USC 2321 et seq.). Unauthorized seedmultiplication is prohibited.

TABLE 1 VARIETY DESCRIPTION INFORMATION - * X00N540 1. TYPE & YIELD:Grain Texture DENT Yield (bushels per acre) 207.57 2. MATURITY: DaysHeat Units Comparative Relative Maturity (CRM) 99 Emergence to 50% ofplants in silk 49 1265 3. PLANT: Value SE Number Plant Height (to flagleaf) (cm) 252.4 24.08 66 Ear Height (to base of top ear node) (cm)117.7 14.26 34 Length of Top Ear Internode (cm) 17.4 0.8 5 Number ofNodes Above Ground 14.4 0.49 5 Anthocyanin of Brace Roots: 1 1 = absent,2 = faint, 3 = moderate, 4 = dark 4. LEAF: Width of Ear Node Leaf (cm)8.2 0.4 5 Length of Ear Node Leaf (cm) 87 2.37 5 Number of Leaves AboveTop Ear 6 0 5 Leaf Angle (Degrees) 28 2.45 5 (at anthesis, 2nd leafabove top ear to the stalk) Leaf Color Very Dark Green Brown Mid Rib(BMR) No Leaf Attitude Semi-erect (appearance of leaf above top ear)Leaf Sheath Pubescence: 1 = none to 9 = like peach fuzz 7 5. TASSEL:Number of Primary Lateral Branches 8.8 0.4 5 Number of SecondaryBranches 1.6 1.02 5 Branch Angle from Central Spike (Degrees) 32 6.78 5Tassel Length: (from peduncle node to tassel tip) (cm) 57.6 1.5 5Peduncle Length: (From top leaf node to lower 18.2 2.04 5 branch) (cm)Central Spike Length (cm) 26.8 1.72 5 Flag Leaf Length (cm) 44.8 1.94 5(from flag leaf collar to tassel tip) Pollen Shed: 0 = male sterile, 9 =heavy shed Anther Color: Salmon Glume Color: Light Green 6a. EAR(Unhusked ear): Silk color: (~3 days after silk emergence) Green-YellowDry husk color: (~65 days after 50% silking) White Husk Tightness: (1 =very loose, 9 = very tight) 3 Husk Extension (at harvest): 1 = short(ears exposed), 2 2 = medium (<8 cm), 3 = long (8-10 cm), 4 = very long(>10 cm) 6b. EAR (Husked ear data): Length of Interior Husk (cm) 18.41.36 5 Shank Length (cm) 8.8 1.17 5 Ear Length (cm) 16.1 1.62 5 EarDiameter at mid-point (mm) 46.5 1.86 5 Ear Weight (gm) 162.2 30.67 5Number of Kernel Rows 17.2 0.98 5 Number of Kernel Per Rows 27.4 4.41 5Kernel Rows: 1 = indistinct, 2 = distinct 2 Row Alignment: 1 1 =straight, 2 = slightly curved, 3 = spiral Ear Taper: 1 1 = slightcylind., 2 = average, 3 = extreme conic. 7. KERNEL (Dried): KernelLength (mm) 12.6 0.59 5 Kernel Width (mm) 7.8 0.59 5 Kernel Thickness(mm) 4.3 0.21 5 Kernel Pericarp color Clear Aleurone Color PatternHomozygous Aleurone Color Clear Hard Endosperm Color Yellow 8. COB: CobDiameter at mid-point (mm) 22.5 0.93 5 Cob Color Red * Wherein X00N540has one or more locus conversion(s) for insect control and/or herbicidetolerance. Number is the number of individual plants that were scored.Value is an average if more than one plant or plot is score.

What is claimed is:
 1. A seed of hybrid maize variety X00N540,representative seed produced by crossing a first plant of variety PH48RSwith a second plant of variety PH42V3, wherein representative seed ofthe varieties PH48RS and PH42V3 have been deposited under ATCC AccessionNumbers PTA-126645 and PTA-124764, respectively.
 2. A plant or plantpart of hybrid maize variety X00N540 grown from the seed of claim 1,wherein the plant part comprises at least one cell of hybrid maizevariety X00N540.
 3. A method of producing the seed of claim 1, themethod comprising crossing a plant of variety PH48RS with a plant ofvariety PH42V3.
 4. The seed of claim 1, further comprising a transgene,wherein the transgene is introduced by backcrossing or genetictransformation into the variety PH48RS, the variety PH42V3, or bothvarieties PH48RS and PH42V3.
 5. A seed of hybrid maize variety X00N540further comprising a single locus conversion, wherein a plant grown fromthe seed comprises a trait conferred by the single locus conversion, andwherein the seed is produced by crossing a first plant of variety PH48RSwith a second plant of variety PH42V3, wherein the first plant, thesecond plant or both further comprise the single locus conversion, andwherein representative seed of the varieties PH48RS and PH42V3 have beendeposited under ATCC Accession Numbers PTA-126645 and PTA-124764,respectively.
 6. The hybrid maize variety X00N540 seed of claim 5,wherein the locus conversion confers a property selected from the groupconsisting of male sterility, herbicide tolerance, insect resistance,disease resistance, waxy starch, modified fatty acid metabolism,modified phytic acid metabolism, modified carbohydrate metabolism andmodified protein metabolism.
 7. The hybrid maize variety X00N540 seed ofclaim 5, further comprising a seed treatment on the surface of the seed.8. A method for producing nucleic acids, the method comprising isolatingnucleic acids from the hybrid maize variety X00N540 seed of claim
 5. 9.A plant or plant part grown from the hybrid maize variety X00N540 seedof claim 5, the plant part comprising at least one cell of hybrid maizevariety X00N540 further comprising the single locus conversion.
 10. Amethod of producing a commodity plant product comprising starch, syrup,silage, fat or protein, the method comprising producing the commodityplant product from the plant or plant part of claim
 9. 11. A method forproducing a second maize plant, the method comprising applying plantbreeding techniques to the plant or plant part of claim 9 to produce thesecond maize plant.
 12. A method for producing a hybrid maize varietyX00N540 seed further comprising a locus conversion, the methodcomprising crossing a first plant of variety PH48RS with a second plantof variety PH42V3, representative seed of the varieties PH48RS andPH42V3 having been deposited under ATCC Accession Numbers PTA-126645 andPTA-124764, respectively, wherein at least one of the varieties PH48RSand PH42V3 further comprises the locus conversion.
 13. An F1 hybridmaize variety X00N540 seed further comprising a locus conversionproduced by the method of claim 12, wherein the seed produces a plantexpressing the traits conferred by the locus conversion and comprisingotherwise all the physiological and morphological characteristics ofmaize variety X00N540 when grown under the same environmentalconditions.
 14. The seed of claim 13, further comprising a seedtreatment on the surface of the seed.
 15. The seed of claim 13, whereinthe locus conversion confers a property selected from the groupconsisting of male sterility, herbicide tolerance, insect resistance,disease resistance, waxy starch, modified fatty acid metabolism,modified phytic acid metabolism, modified carbohydrate metabolism andmodified protein metabolism.
 16. A method for producing nucleic acids,the method comprising isolating nucleic acids from the seed of claim 13.17. A plant or plant part produced by growing the seed of claim 13, theplant part comprising at least one F1 hybrid maize variety X00N540 cellfurther comprising the locus conversion.
 18. A method for producingnucleic acids, the method comprising isolating nucleic acids from theplant or plant part of claim
 17. 19. A method of producing a commodityplant product comprising starch, syrup, silage, fat or protein, themethod comprising producing the commodity plant product from the plantor plant part of claim
 17. 20. A method for producing a second maizeplant, the method comprising crossing the maize plant or plant part ofclaim 17 with itself or with a different maize plant.